Detection of Deformed Wing Virus (DWV) in honeybee (Apis mellifera L.) and Varroa mites (Varroa destructor) and Kakugo Virus (KV) in aggressive honeybees using RT-PCR in Egypt
The identification of bee viruses is of considerable importance particularly when considering the lack of information on the natural incidence of virus infection in honeybee population Worldwide. All samples evaluated in this study were from honeybee (Apis mellifera L.) collected from Giza Governorate, Egypt. Total RNA was isolated from thorax, abdomen of asymptomatic, deformed wing honeybees, head of aggressive and deformed wing honeybees and Varroa mites.Two fragments of 250 and 540 bp of deformed wing virus (DWV) and Kakugo Virus (KV) , respectively, were amplified using conventional RT-PCR, and were sequenced and analyzed. When analyzing head, thorax and abdomen of crippled bees all body parts were always strongly positive DWV sequence in contrast, some asymptomatic bees viral sequences could not be detected in RNAs extracted from all body parts. Analysis of mites (group of 50 mites) revealed the presence of DWV. Interestingly, DWV virus replication was correlated with wing deformity, while KV was detected specifically in the head of aggressive worker honeybees but not in the thorax or abdomen. Viral detection and identification was confirmed by sequencing of the PCR products using conventional RT-PCR. Furthermore, virus titers in different body parts were compared by real-time RT-PCR to identify potential tissue reservoirs of virus replication in the body of honeybees. Homology search of DWV PCR amplified fragment revealed similarities of 99% with DWV isolate Warwick-2009 poly-protein gene (accession number: GU109335.1) and 98% with DWV isolate Chilensis A complete genome (accession number: JQ413340.1). The genetic distance between the nucleotide sequence of the amplified PCR product of DWV detected in honeybees in Egypt, Warwick isolate and Chilensis isolate was 0.020 and 0.025, respectively. Also, homology search of KV PCR amplified fragment revealed similarities of 97% with KV isolate KV_Ox genomic sequence (accession number: KC786224.1), 90.9% with DWV_Ox genomic sequence (accession number: KC786223.1) and 94.4% with DWV isolate Warwick-2009 poly-protein gene (accession number: GU109335.1), the genetic distance between the nucleotide sequence of the amplified PCR product of KV detected in honeybees in Egypt KV_Ox genomic sequence and DWV_Ox genomic sequence was 0.019. The described real-time SG RT-PCR proved to be a fast, accurate and useful technique to detect and quantify low viral loads that cause unapparent infections and might contribute with other factors to the increasing honeybee colonies depopulation.
