**2. Results**

Collected samples of bumblebees, originating from different locations in Croatia, were found positive with prevalence for ABPV (3.70%), BQCV (88.89%), CBPV (3.70%), and DWV (37.40%) (Figure 1).

**Figure 1.** The determined prevalence (%) of the 27 tested bumblebee samples for the presence of four different honeybee viruses (ABPV, BQCV, CBPV, and DWV).

The obtained Ct value for one ABPV-positive sample was 35.04, corresponding to 9.082 × 102 copy number/5 <sup>μ</sup>L. Altogether, BQCV was detected in 24 out of 27 samples, with Ct values from the lowest 17.24 to the highest 29.71 (corresponding to copy number/5 <sup>μ</sup>L from 1.251 × 104 to 1.224 × 108). One bumblebee sample was detected as CBPV-positive with Ct value 41.00. The detected Ct values for ten DWV-positive samples varied from 27.38 to 41.84 with copy number/5 <sup>μ</sup>L from 6.33 to 6.392 × <sup>10</sup><sup>4</sup> (Table 1).


**Table 1.** The determined copy number in 27 tested bumblebee samples by real-time RT-PCR assay for the detection of ABPV, BQCV, CBPV, and DWV, collected from 27 different geographical locations in Croatia.

> For 24 BQCV-positive bumblebee samples, the 653 nucleotide long sequences were successfully determined and compared with those available in GenBank. High genetic diversity among 24 Croatian sequenced BQCV-positive samples in bumblebee were identified, with 95.7 to 100% nucleotide identity to each other. The majority of the identified BQCV strains were closely related with BQCV stains from Slovenia. Strain BQCV Bombus-Gos/2018 (MW488258) has 100% nucleotide identity with bumblebee strain BQCV Bombus BT23/2017 (MH900014) and honeybee isolate 279/2017 (MH899977), both detected in the neighboring country of Slovenia. A group of 17 closely related BQCV-positive samples, located on the same branch of phylogenetic tree, were collected throughout the territory of Croatia, and have from 98.47 to 100% nucleotide identity with isolate 281/2017 (MH899979), detected in Slovenia. The BQCV strain Bombus-Var/2018 (MW488242) has 99.54% nucleotide identities with isolate BQCV 637/2009 (MH899994) detected in Slovenia and 98.82% nucleotide identity with strain Sydney ((MF623171) detected in Australia. Strain Bombus-Vet/2018 (MW488256) has 99.69% nucleotide identity with strain 287-1/2007

(MH899983), while with the strain Bombus-Mak/2018 (MW488239) has 99.08% identity. Detected strain Bombus-Kri/2018 (MW488238) collected in Križevci showed 100% nucleotide identity with Slovenian honeybee strain 1/2008 (MH899946). Strain Bombus-Ses/2018 (MW488243) and Bombus-Nez/2018 (MW488260) have 99.39% nucleotide identities with isolate 1956/2009 (MH899996) identified in Slovenia in 2009 in clinically affected honeybee samples (Figure 2).

**Figure 2.** The BQCV tree was constructed from 653 nt long sequences of capsid protein gene (between nt positions 7774 and 8426; numbering according to BQCV isolate Sydney, MF623171) for 24 Croatian and 56 DWV isolates from GenBank. BQCV bumblebee samples from Croatia (•) and BQCV bumblebee positive samples from Genbank (O) are marked on the phylogenetic tree. The maximum likelihood phylogenetic tree with the Tamura 3-parameter substitution model with Gamma distribution. Statistical support for the tree was evaluated by bootstrapping, based on 1000 repetitions. Bootstrap values lower than 70 are not shown.

For 2 samples out of 10 DWV-positive bumblebee samples, the 471 nucleotide long sequences were successfully determined and compared with those available in GenBank. A DWV-positive sample Bombus-Mir/2018 (MW488261) was collected in 2018 in Zagreb and has 98.09% nucleotide identity with the closely related strains YU4 (JF346630) and YU5 (JF346631) in GenBank, collected in 1988 near Turopoljski Lug, which is located about 50 km from Zagreb city. The second DWV-positive sample Bombus-Nik/2018 (MW488262) was collected near Šibenik and has 98.09% nucleotide identity with strain SLO/BM199/2013, detected in *A. mellifera* in 2013 in Slovenia, while with DWV strain MeDWV1 (MW222481) and DWV strain Maryland/2015/422 (MG831202), both from the USA, it shares from 97.45 to 97.66% nucleotide identity (Figure 3).

**Figure 3.** The DWV tree was constructed for two Croatian and 33 DWV isolates from GenBank from 471 nt long sequences of L protein gene (between nt positions 1357 and 1827; numbering according to isolate Austria 1414, KU847397). The two DWV bumblebee samples from Croatia determined in this study are marked (•) on the phylogenetic tree. The maximum likelihood phylogenetic tree with the Tamura 3-parameter substitution model with Gamma distribution. Statistical support for the tree was evaluated by bootstrapping, based on 1000 repetitions. Bootstrap values lower than 70 are not shown.

Genetically closely related strains, previously detected in the same geographic region among honeybees, were also detected in randomly selected bumblebee samples in Croatia. Twenty-six determined sequences from this study were deposited in GenBank with accession numbers MW488237-MW488262.
