Dataset Information

Rapid detection of deformed wing virus in honeybee using ultra-rapid qPCR and a DNA-chip.

ABSTRACT: Fast and accurate detection of viral RNA pathogens is important in apiculture. A polymerase chain reaction (PCR)-based detection method has been developed, which is simple, specific, and sensitive. In this study, we rapidly (in 1 min) synthesized cDNA from the RNA of deformed wing virus (DWV)-infected bees (Apis mellifera), and then, within 10 min, amplified the target cDNA by ultra-rapid qPCR. The PCR products were hybridized to a DNA-chip for confirmation of target gene specificity. The results of this study suggest that our method might be a useful tool for detecting DWV, as well as for the diagnosis of RNA virus-mediated diseases on-site.

SUBMITTER: Kim JM

PROVIDER: S-EPMC7000893 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rapid detection of deformed wing virus in honeybee using ultra-rapid qPCR and a DNA-chip.

Kim Jung Min JM Lim Su Jin SJ Kim SoMin S Kim MoonJung M Kim ByoungHee B Tai Truong A TA Kim Seonmi S Yoon ByoungSu B

Journal of veterinary science 20200101 1

Fast and accurate detection of viral RNA pathogens is important in apiculture. A polymerase chain reaction (PCR)-based detection method has been developed, which is simple, specific, and sensitive. In this study, we rapidly (in 1 min) synthesized cDNA from the RNA of deformed wing virus (DWV)-infected bees (<i>Apis mellifera</i>), and then, within 10 min, amplified the target cDNA by ultra-rapid qPCR. The PCR products were hybridized to a DNA-chip for confirmation of target gene specificity. The ...[more]

PMID: 31940683

Similar Datasets

Project description:BackgroundDeformed wing virus (DWV) is one of many viruses that infect honeybees and has been extensively studied because of its close association with honeybee colony collapse that is induced by Varroa destructor. However, virus genotypes, sequence characteristics, and genetic variations of DWV remain unknown in China.MethodsTwo DWV strains were isolated from Jinzhou and Qinhuangdao cities in China, and were named China1-2017 (accession number: MF770715) and China2-2018 (accession number: MH165180), respectively, and their complete genome sequences were analyzed. To investigate the phylogenetic relationships of the DWV isolates, a phylogenetic tree of the complete open reading frame (ORF), structural protein VP1, and non-structural protein 3C+RdRp of the DWV sequences was constructed using the MEGA 5.0 software program. Then, the similarity and recombinant events of the DWV isolated strains were analyzed using recombination detection program (RDP4) software and genetic algorithm for recombination detection (GARD).ResultsThe complete genomic analysis showed that the genomes of the China1-2017 and China2-2018 DWV strains consisted of 10,141 base pairs (bp) and 10,105 bp, respectively, and contained a single, large ORF (China1-2017: 1,146-9,827 bp; China2-2018: 1,351-9,816 bp) that encoded 2,894 amino acids. The sequences were compared with 20 previously reported DWV sequences from different countries and with sequences of two closely related viruses, Kakugo virus (KV) and V. destructor virus-1. Multiple sequence comparisons revealed a nucleotide identity of 84.3-96.7%, and identity of 94.7-98.6% in amino acids between the two isolate strains and 20 reference strains. The two novel isolates showed 96.7% nucleotide identity and 98.1% amino acid identity. The phylogenetic analyses showed that the two isolates belonged to DWV Type A and were closely related to the KV-2001 strain from Japan. Based on the RDP4 and GARD analyses, the recombination of the China2-2018 strain was located at the 4,266-7,507 nt region, with Korea I-2012 as an infer unknown parent and China-2017 as a minor parent, which spanned the entire helicase ORF. To the best of our knowledge, this is the first study to the complete sequence of DWV isolated from Apis cerana and the possible DWV recombination events in China. Our findings are important for further research of the phylogenetic relationship of DWVs in China with DWV strains from other countries and also contribute to the understanding of virological properties of these complex DWV recombinants.

Project description:Sacbrood virus (SBV) and Deformed wing virus (DWV) are evolutionarily related positive-strand RNA viruses, members of the Iflavirus group. They both infect the honeybee Apis mellifera but have strikingly different levels of virulence when transmitted orally. Honeybee larvae orally infected with SBV usually accumulate high levels of the virus, which halts larval development and causes insect death. In contrast, oral DWV infection at the larval stage usually causes asymptomatic infection with low levels of the virus, although high doses of ingested DWV could lead to DWV replicating to high levels. We investigated effects of DWV and SBV infection on the transcriptome of honeybee larvae and pupae using global RNA-Seq and real-time PCR analysis. This showed that high levels of SBV replication resulted in down-regulation of the genes involved in cuticle and muscle development, together with changes in expression of putative immune-related genes. In particular, honeybee larvae with high levels of SBV replication, with and without high levels of DWV replication, showed concerted up-regulated expression of antimicrobial peptides (AMPs), and down-regulated expression of the prophenoloxidase activating enzyme (PPAE) together with up-regulation of the expression of a putative serpin, which could lead to the suppression of the melanisation pathway. The effects of high SBV levels on expression of these immune genes were unlikely to be a consequence of SBV-induced developmental changes, because similar effects were observed in honeybee pupae infected by injection. In the orally infected larvae with high levels of DWV replication alone we observed no changes of AMPs or of gene expression in the melanisation pathway. In the injected pupae, high levels of DWV alone did not alter expression of the tested melanisation pathway genes, but resulted in up-regulation of the AMPs, which could be attributed to the effect of DWV on the regulation of AMP expression in response to wounding. We propose that the difference in expression of the honeybee immune genes induced by SBV and DWV may be an evolutionary adaptation to the different predominant transmission routes used by these viruses.

Dataset Information

Rapid detection of deformed wing virus in honeybee using ultra-rapid qPCR and a DNA-chip.

Publications

Rapid detection of deformed wing virus in honeybee using ultra-rapid qPCR and a DNA-chip.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure