The ectoparasitic mite Varroa destructor transmits and triggers viral infections that have deleterious effects on honey bee colonies worldwide. We performed a manipulative experiment in which worker bees collected at emergence were exposed to Varroa for 72 h, and their proteomes were compared with those of untreated control bees. Label-free quantitative proteomics identified 77 differentially expressed A. mellifera proteins (DEPs). In addition, viral proteins were identified by orthogonal analysis, and most importantly, Deformed wing virus (DWV) was found at high levels/intensity in Varroa-exposed bees. Pathway enrichment analysis suggested that the main pathways affected included peroxisomal metabolism, cyto-/exoskeleton reorganization, and cuticular proteins. Detailed examination of individual DEPs revealed that additional changes in DEPs were associated with peroxisomal function. In addition, the proteome data support the importance of TGF-β signaling in Varroa-DWV interaction and the involvement of the mTORC1 and Hippo pathways. These results suggest that the effect of DWV on bees associated with Varroa feeding results in aberrant autophagy. In particular, autophagy is selectively modulated by peroxisomes, to which the observed proteome changes strongly corresponded. This study complements previous research with different study designs and suggests the importance of the peroxisome, which plays a key role in viral infections.
- Klíčová slova
- Apis mellifera, DWV, autophagy, host‐pathogen interaction, lipid metabolism,
- MeSH
- hmyzí proteiny metabolismus MeSH
- interakce hostitele a parazita MeSH
- peroxizomy * metabolismus virologie MeSH
- proteom metabolismus analýza MeSH
- proteomika metody MeSH
- RNA-viry * fyziologie MeSH
- signální transdukce MeSH
- Varroidae * virologie MeSH
- včely virologie parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- hmyzí proteiny MeSH
- proteom MeSH
The western honeybee is the primary pollinator of numerous food crops. Furthermore, honeybees are essential for ecosystem stability by sustaining the diversity and abundance of wild flowering plants. However, the worldwide population of honeybees is under pressure from environmental stress and pathogens. Viruses from the families Iflaviridae and Dicistroviridae, together with their vector, the parasitic mite Varroa destructor, are the major threat to the world's honeybees. Dicistroviruses and iflaviruses have capsids with icosahedral symmetries. Acidic pH triggers the genome release of both dicistroviruses and iflaviruses. The capsids of iflaviruses expand, whereas those of dicistroviruses remain compact until the genome release. Furthermore, dicistroviruses use inner capsid proteins, whereas iflaviruses employ protruding domains or minor capsid proteins from the virion surface to penetrate membranes and deliver their genomes into the cell cytoplasm. The structural characterization of the infection process opens up possibilities for the development of antiviral compounds.
- MeSH
- genom virový * MeSH
- kapsida chemie metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- kyseliny MeSH
- molekulární modely MeSH
- RNA-viry metabolismus MeSH
- včely virologie MeSH
- virion chemie genetika MeSH
- virové nemoci veterinární MeSH
- virové plášťové proteiny chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- kyseliny MeSH
- virové plášťové proteiny MeSH
Among the many diseases compromising the well-being of the honey bee (Apis mellifera) the chronic paralysis syndrome of adult honey bees is one of the best described. The causative agent, chronic bee paralysis virus (CBPV), is a positive sense, single-stranded RNA virus with a segmented genome. Segment 1 encodes three putative open reading frames (ORFs), including the RNA-dependent RNA polymerase and other non-structural protein coding regions. Segment 2 encodes four putative ORFs, which contain the genes of supposed structural proteins. In this study, we established a reverse genetic system for CBPV by molecular cloning of DNA copies of both genome segments. CBPV rescue was studied in imago and honey bee pupae infection models. Virus replication and progeny virus production was only initiated when capped RNAs of both genome segments were injected in honey bees. As injection of these clonal RNAs caused clinical symptoms similar to wild-type CBPV infection, we conclude that the novel molecular clone fulfilled Koch's postulates. Our virus clone will enable in-depth analysis of CBPV pathogenesis and help to increase knowledge about this important honey bee disease.
Honeybee workers undergo metamorphosis in capped cells for approximately 13 days before adult emergence. During the same period, Varroa mites prick the defenseless host many times. We sought to identify proteome differences between emerging Varroa-parasitized and parasite-free honeybees showing the presence or absence of clinical signs of deformed wing virus (DWV) in the capped cells. A label-free proteomic analysis utilizing nanoLC coupled with an Orbitrap Fusion Tribrid mass spectrometer provided a quantitative comparison of 2316 protein hits. Redundancy analysis (RDA) showed that the combination of Varroa parasitism and DWV clinical signs caused proteome changes that occurred in the same direction as those of Varroa alone and were approximately two-fold higher. Furthermore, proteome changes associated with DWV signs alone were positioned above Varroa in the RDA. Multiple markers indicate that Varroa activates TGF-β-induced pathways to suppress wound healing and the immune response and that the collective action of stressors intensifies these effects. Furthermore, we indicate JAK/STAT hyperactivation, p53-BCL-6 feedback loop disruption, Wnt pathway activation, Wnt/Hippo crosstalk disruption, and NF-κB and JAK/STAT signaling conflict in the Varroa-honeybee-DWV interaction. These results illustrate the higher effect of Varroa than of DWV at the time of emergence. Markers for future research are provided.
- MeSH
- biologické markery MeSH
- biologické modely MeSH
- histony metabolismus MeSH
- Janus kinasy metabolismus MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- proteiny Wnt metabolismus MeSH
- proteom * MeSH
- proteomika * MeSH
- reaktivní formy kyslíku metabolismus MeSH
- RNA-viry * MeSH
- signální transdukce MeSH
- symbióza * MeSH
- transformující růstový faktor beta * MeSH
- transkripční faktory STAT metabolismus MeSH
- Varroidae * MeSH
- včely metabolismus parazitologie virologie MeSH
- výpočetní biologie metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- biologické markery MeSH
- histony MeSH
- Janus kinasy MeSH
- protein-serin-threoninkinasy MeSH
- proteiny Wnt MeSH
- proteom * MeSH
- reaktivní formy kyslíku MeSH
- transformující růstový faktor beta * MeSH
- transkripční faktory STAT MeSH
Instrumental insemination of Apis mellifera L. queens is a widely employed technique used in honeybee breeding that enables the effective control of mating. However, drone semen represents a potential source of honeybee viruses. In this study, 43 semen doses collected from apparently healthy drones, and consequently used in instrumental insemination, were analysed using PCR or RT-PCR to detect the presence of viral genome of 11 honeybee viruses. In 91% of samples, viral infection was detected. The survey revealed genomes of five viruses, namely Deformed wing virus (DWV), Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Sacbrood virus (SBV), and A. mellifera filamentous virus (AmFV) in 84%, 19%, 14%, 2%, and 67% of samples, respectively. Single infection (30% of samples) as well as multiple infection (61% of samples) of two, three or four pathogens were also evaluated. To the best of our knowledge, this is the first study describing the presence of the BQCV and SBV genome sequence in drone ejaculate. Phylogenetic analysis of BQCV partial helicase gene sequence revealed the high similarity of nucleotide sequence of described Czech strains, which varied from 91.4% to 99.6%. The findings of our study indicate the possibility of venereal transmission of BQCV and SBV.
- Klíčová slova
- BQCV, SBV, ejaculate, honeybees, instrumental insemination, virus detection,
- MeSH
- biodiverzita * MeSH
- chov metody MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- polymerázová řetězová reakce MeSH
- sperma virologie MeSH
- včely virologie MeSH
- viry klasifikace genetika izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Infection by sacbrood virus (SBV) from the family Iflaviridae is lethal to honey bee larvae but only rarely causes the collapse of honey bee colonies. Despite the negative effect of SBV on honey bees, the structure of its particles and mechanism of its genome delivery are unknown. Here we present the crystal structure of SBV virion and show that it contains 60 copies of a minor capsid protein (MiCP) attached to the virion surface. No similar MiCPs have been previously reported in any of the related viruses from the order Picornavirales. The location of the MiCP coding sequence within the SBV genome indicates that the MiCP evolved from a C-terminal extension of a major capsid protein by the introduction of a cleavage site for a virus protease. The exposure of SBV to acidic pH, which the virus likely encounters during cell entry, induces the formation of pores at threefold and fivefold axes of the capsid that are 7 Å and 12 Å in diameter, respectively. This is in contrast to vertebrate picornaviruses, in which the pores along twofold icosahedral symmetry axes are currently considered the most likely sites for genome release. SBV virions lack VP4 subunits that facilitate the genome delivery of many related dicistroviruses and picornaviruses. MiCP subunits induce liposome disruption in vitro, indicating that they are functional analogs of VP4 subunits and enable the virus genome to escape across the endosome membrane into the cell cytoplasm.
- Klíčová slova
- genome, honeybee, release, structure, virus,
- MeSH
- endozomy chemie metabolismus virologie MeSH
- genom virový * MeSH
- krystalografie rentgenová MeSH
- RNA-viry * chemie metabolismus MeSH
- včely virologie MeSH
- virion * chemie metabolismus MeSH
- virové plášťové proteiny * chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- virové plášťové proteiny * MeSH
BACKGROUND: Honeybee viruses have been recognized as being among the most important factors leading to colony losses worldwide. Colony food and faeces are regarded as possible sources of infectious viruses able to contaminate the environment and equipment of apiaries. Thus, methods for elimination of viruses are required. No cell culture assay for testing the effect of disinfectants on honeybee viruses is yet available. Therefore, surrogate virus was employed for testing of the efficacy of iodophor- and peracetic acid-based disinfectants in combination with six organic contaminants at +6 °C and +22 °C. Moreover, we evaluated the persistence of the surrogate in honey at +6 °C, +22 °C, and +50 °C. RESULTS: Iodophor-based disinfectant showed a maximum reduction of virus titre of 3.4 log10 . Peracetic acid reduced the titre (≥4 log10 ) only at 22 °C and without yeast extract/bovine serum albumin. After 25 days of incubation of the virus - honey mix, no decrease of virus titre was observed at +6 °C, whereas a significant reduction (3.5 log10 ) was found at +50 °C already after 1 day. CONCLUSIONS: Both tested disinfectants can serve as appropriate virucides in apiaries. The effect of peracetic acid significantly depended on temperature and organic contaminants. The iodophor-based disinfectant showed a stable antiviral effect at different temperatures and with different contaminants. © 2017 Society of Chemical Industry.
- Klíčová slova
- beekeeping, enterovirus, iodophor, peracetic acid, stability, surface, virucidal efficacy,
- MeSH
- antivirové látky farmakologie MeSH
- dezinficiencia farmakologie MeSH
- Enterovirus účinky léků fyziologie MeSH
- včelařství MeSH
- včely fyziologie virologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antivirové látky MeSH
- dezinficiencia MeSH
The worldwide population of western honey bees (Apis mellifera) is under pressure from habitat loss, environmental stress, and pathogens, particularly viruses that cause lethal epidemics. Deformed wing virus (DWV) from the family Iflaviridae, together with its vector, the mite Varroa destructor, is likely the major threat to the world's honey bees. However, lack of knowledge of the atomic structures of iflaviruses has hindered the development of effective treatments against them. Here, we present the virion structures of DWV determined to a resolution of 3.1 Å using cryo-electron microscopy and 3.8 Å by X-ray crystallography. The C-terminal extension of capsid protein VP3 folds into a globular protruding (P) domain, exposed on the virion surface. The P domain contains an Asp-His-Ser catalytic triad that is, together with five residues that are spatially close, conserved among iflaviruses. These residues may participate in receptor binding or provide the protease, lipase, or esterase activity required for entry of the virus into a host cell. Furthermore, nucleotides of the DWV RNA genome interact with VP3 subunits. The capsid protein residues involved in the RNA binding are conserved among honey bee iflaviruses, suggesting a putative role of the genome in stabilizing the virion or facilitating capsid assembly. Identifying the RNA-binding and putative catalytic sites within the DWV virion structure enables future analyses of how DWV and other iflaviruses infect insect cells and also opens up possibilities for the development of antiviral treatments.
- Klíčová slova
- Apis mellifera, colony collapse disorder, honey bee, structure, virus,
- MeSH
- elektronová kryomikroskopie MeSH
- kapsida ultrastruktura MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- počítačové zpracování obrazu MeSH
- proteinové domény MeSH
- RNA-viry ultrastruktura MeSH
- sekvence aminokyselin MeSH
- včely virologie MeSH
- virion ultrastruktura MeSH
- virové plášťové proteiny chemie ultrastruktura MeSH
- viry hmyzu ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- virové plášťové proteiny MeSH
Viral diseases are a major threat to honeybee (Apis mellifera) populations worldwide and therefore an important factor in reliable crop pollination and food security. Black queen cell virus (BQCV) is the etiological agent of a fatal disease of honeybee queen larvae and pupae. The virus belongs to the genus Triatovirus from the family Dicistroviridae, which is part of the order Picornavirales Here we present a crystal structure of BQCV determined to a resolution of 3.4 Å. The virion is formed by 60 copies of each of the major capsid proteins VP1, VP2, and VP3; however, there is no density corresponding to a 75-residue-long minor capsid protein VP4 encoded by the BQCV genome. We show that the VP4 subunits are present in the crystallized virions that are infectious. This aspect of the BQCV virion is similar to that of the previously characterized triatoma virus and supports the recent establishment of the separate genus Triatovirus within the family Dicistroviridae The C terminus of VP1 and CD loops of capsid proteins VP1 and VP3 of BQCV form 34-Å-tall finger-like protrusions at the virion surface. The protrusions are larger than those of related dicistroviruses.IMPORTANCE The western honeybee is the most important pollinator of all, and it is required to sustain the agricultural production and biodiversity of wild flowering plants. However, honeybee populations worldwide are suffering from virus infections that cause colony losses. One of the most common, and least known, honeybee pathogens is black queen cell virus (BQCV), which at high titers causes queen larvae and pupae to turn black and die. Here we present the three-dimensional virion structure of BQCV, determined by X-ray crystallography. The structure of BQCV reveals large protrusions on the virion surface. Capsid protein VP1 of BQCV does not contain a hydrophobic pocket. Therefore, the BQCV virion structure provides evidence that capsid-binding antiviral compounds that can prevent the replication of vertebrate picornaviruses may be ineffective against honeybee virus infections.
- Klíčová slova
- Apis mellifera, Cripavirus, Dicistroviridae, Picornavirales, Triatovirus, X ray, X-ray crystallography, capsid, crystallography, honey bee, honeybee, insect disease, structure, virion, virus,
- MeSH
- Dicistroviridae ultrastruktura MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- včely virologie MeSH
- virion ultrastruktura MeSH
- virové plášťové proteiny chemie MeSH
- virové struktury MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- virové plášťové proteiny MeSH
UNLABELLED: Viruses of the family Dicistroviridae can cause substantial economic damage by infecting agriculturally important insects. Israeli acute bee paralysis virus (IAPV) causes honeybee colony collapse disorder in the United States. High-resolution molecular details of the genome delivery mechanism of dicistroviruses are unknown. Here we present a cryo-electron microscopy analysis of IAPV virions induced to release their genomes in vitro We determined structures of full IAPV virions primed to release their genomes to a resolution of 3.3 Å and of empty capsids to a resolution of 3.9 Å. We show that IAPV does not form expanded A particles before genome release as in the case of related enteroviruses of the family Picornaviridae The structural changes observed in the empty IAPV particles include detachment of the VP4 minor capsid proteins from the inner face of the capsid and partial loss of the structure of the N-terminal arms of the VP2 capsid proteins. Unlike the case for many picornaviruses, the empty particles of IAPV are not expanded relative to the native virions and do not contain pores in their capsids that might serve as channels for genome release. Therefore, rearrangement of a unique region of the capsid is probably required for IAPV genome release. IMPORTANCE: Honeybee populations in Europe and North America are declining due to pressure from pathogens, including viruses. Israeli acute bee paralysis virus (IAPV), a member of the family Dicistroviridae, causes honeybee colony collapse disorder in the United States. The delivery of virus genomes into host cells is necessary for the initiation of infection. Here we present a structural cryo-electron microscopy analysis of IAPV particles induced to release their genomes. We show that genome release is not preceded by an expansion of IAPV virions as in the case of related picornaviruses that infect vertebrates. Furthermore, minor capsid proteins detach from the capsid upon genome release. The genome leaves behind empty particles that have compact protein shells.
- Klíčová slova
- Aparavirus, Apis mellifera, CCD, Dicistroviridae, Picornavirales, capsid, colony collapse disorder, cryo, electron microscopy, empty, genome, honey bee, honeybee, release, structure, uncoating, virion, virus,
- MeSH
- Dicistroviridae fyziologie ultrastruktura MeSH
- elektronová kryomikroskopie * MeSH
- genom virový * MeSH
- kapsida metabolismus MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- sestavení viru MeSH
- svlékání virového obalu * MeSH
- včely virologie MeSH
- virion fyziologie ultrastruktura MeSH
- virové plášťové proteiny chemie genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- virové plášťové proteiny MeSH
