Viral diseases of honeybees are a major problem in apiculture, causing serious economic losses worldwide, especially in combination with varroa mites. To increase understanding of the relationship among viruses, mites and colony decline, the tripartite relationships among bees, viruses [Kashmir bee virus (KBV) and sacbrood virus (SBV)] and varroa mites have been investigated systematically. To develop an antibody-based test for KBV, two structural recombinant proteins were purified for polyclonal-antibody production. By using ELISA and RT-PCR, the presence of KBV and SBV was studied comparatively in different developmental stages and castes of bees. The results demonstrated that KBV may persist as a viral genome with extremely low levels of viral-capsid proteins and that KBV and SBV can co-infect honeybees. This study indicated the presence of KBV and SBV RNAs in both queens and eggs by RT-PCR, suggesting a route of transovarial transmission. Horizontal transmission is also very likely among adult bees and from adult workers to larvae through contaminated food resources, because both viruses have been detected in all developmental stages and food sources (brood food, honey, pollen and royal jelly). Furthermore, it was demonstrated that mites were another possible route of horizontal transmission, as both viruses were detected in mites and their saliva. This study, for the first time, detected co-occurrence of viruses in varroa, further underlining the importance of the mites in vectoring different bee viruses. Therefore, these results indicated that multiple infection routes exist for honeybee viral diseases.
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