*Article* **The First Detection and Genetic Characterization of Four Different Honeybee Viruses in Wild Bumblebees from Croatia**

**Ivana Tlak Gajger 1,\*, Laura Šimenc <sup>2</sup> and Ivan Toplak <sup>2</sup>**


**\*** Correspondence: ivana.tlak@vef.hr; Tel.: +385-91-2390-041

**Abstract:** To determine the presence and the prevalence of four different honeybee viruses (acute bee paralysis virus—ABPV, black queen cell virus—BQCV, chronic bee paralysis virus—CBPV, deformed wing virus—DWV) in wild bumblebees, pooled randomly selected bumblebee samples were collected from twenty-seven different locations in the territory of Croatia. All samples were prepared and examined using the RT-PCR methods for quantification of mentioned honeybee viruses. Determined prevalence (%) of identified positive viruses were in the following decreasing order: BQCV > DWV > ABPV, CBPV. Additionally, direct sequencing of samples positive for BQCV (*n* = 24) and DWV (*n* = 2) was performed, as well as a test of molecular phylogeny comparison with those available in GenBank. Selected positive field viruses' strains showed 95.7 to 100% (BQCV) and 98.09% (DWV) nucleotide identity with previously detected and deposited honeybee virus strains in the geographic areas in Croatia and neighboring Slovenia. In this article, the first detection of four honeybee viruses with genetic characterization of high diversity strains circulating in wild bumblebees in Croatia is presented.

**Keywords:** *Bombus* spp.; honeybee viruses; deformed wing virus; black queen cell virus; acute bee paralysis virus; chronic bee paralysis virus; genetic characterization; sequencing; transmission routes
