organism: Deformed wing virus
description: Protocols: Deformed wing virus was replicating in its host, honeybee (Apis mellifra). Bees were developing in a brood frame Varroa-free colony for 3 days after hatching from egg. Then the entire brood frame with bee larvae was transferred to a Varroa-infested colony with high levels of Deformed wing virus (DWV). Both Varroa-free colony and Varroa-infested colony were kept in outdoor flight cages, bees were fed with pollen sourced in Varroa-free region and with sugar syrup. Deformed wing virus (DWV) was produced in its honeybee (Apis mellifera) host subjected to the following treatments. Larvae of Apis mellifra workers (day 3 of bee development), which were hatched from the eggs laid in a colony free from Varroa destructor mites and harbouring low levels of DWV, were either left to develop in the same Varroa-free hive before sampling at pupal stage (day 15) (EnvironmentalHistory- NoTransfer), or were transferred to a Varroa-infested colony with high levels of DWV in bees and the mites (EnvironmentalHistory- TransferToVarroaColony). All transferred larvae were receiving the DWV strains present in this Varroa-infested colony with the food delivered by the nurse bees until capping on day 9. About half of these larvae were capped with Varroa mite and were subjected to the mite piercing and feeding on their haemolymph for 5 days during pupal development until sampling on day 14 (GrowthCondition - MiteFeeding), the rest of the pupae developed without the mite (GrowthCondition - NoMite). Exposure to the mite piercing and feeding resulted in about 1000-fold increase of the DWV levels, overt infection, in about 80% of the mite-exposed pupae (DiseaseState- HighDWV). Preparations of DWV were isolated from Varroa-free control pupae (Virus-prep-Group C), Varroa-free transferred pupae (Virus-prep-Group NV), and Varroa exposed overtly infected pupae (Virus-prep-Group VH). The DWV preparations were injected to haemolymph of the Varroa-free bee pupae, day 11 of bee development, (GrowthCondition - InjectVirusC, - InjectVirusC and - InjectVirusVH, respectively). The injected pupae were incubated in high humidity at +35C for four days (EnvironmentalHistory- Incubator) before development of overt DWV infection (DiseaseState - HighDWV), at this stage total RNA was extracted from the infected pupae. A series of the DWV cDNA fragment covering complete DWV genomic RNA sequences were amplified by RT-PCR using RNA extracted from all virus preparations and the RNA extracts from the injected pupae. The cDNA preparations were sequenced using next generation paired-end sequencing to obtain data on the DWV strain composition. TRIzol reagent (Invitrogen) extraction of total RNA from whole pupae pools and from the virus preparations A series of seven overlapping RT-PCR fragments, ranging from 1.5 to 2.5 kb covering entire DWV genomic RNA sequence, were generated for each the RNA extracts. The cDNA samples were used for library construction using Illumina DNA protocol.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2013-09-01
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2013-05-21
attributes.tag: External Id
attributes.value: SAMEA2073854
attributes.tag: INSDC center alias
attributes.value: School of Life Sciences
attributes.tag: INSDC center name
attributes.value: School of Life Sciences
attributes.tag: INSDC first public
attributes.value: 2013-09-01T17:01:52Z
attributes.tag: INSDC last update
attributes.value: 2013-05-21T14:41:18Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-1675:Virus-prep-Group NV
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: growth condition
attributes.value: NoMite
attributes.tag: host
attributes.value: Apis mellifera
attributes.tag: individual
attributes.value: Virus-prep-Group NV
attributes.tag: sample name
attributes.value: E-MTAB-1675:Virus-prep-Group NV