**2. Results**

The investigated samples, coming from the same honey bee colony, tested positive for *C. mellificae*, *L. passim,* KBV, and DWV (Table 1). No amplicons were detected for *P. larvae, M. plutonious*, ABPV, IAPV, BQCV, SBV, CBPV, SPV major, and SPV minor in SHB individuals and the pool of SHBs.

**Table 1.** Summary of the *Aethina tumida* (SHB = small hive beetle) samples that tested positive for a given pathogen with the RT-PCR.


POS: positive; POS \*: positive samples with replicative virus forms.

One of the SHB individuals was negative for all pathogens, whereas the other nine tested positive for one or two of them. The SHB pool was positive for both trypanosomatid species and the two virus types.

In the SHB individuals, no significant difference was found in the prevalence between *C. mellificae* and *L. passim* positives (bilateral Fisher's exact test: *p* = 0.675). No co-infections with the two were detected.

The frequencies of DWV- and KBV-positive individuals did not significantly differ (bilateral Fisher's exact test: *p* = 0.070). Viral coinfections were found only in one individual SHB, representing a significantly lower proportion of the positives (bilateral Fisher's exact test: *p* = 0.010).

A strand-specific PCR demonstrated active viral replication of KBV and DWV in PCR-positive samples. Blast analysis on the sequences obtained from positive amplicons confirmed the specificity of the results, with high similarity (99%) to specific virus genome sequences deposited in GenBank. For each virus, the same sequence was recorded in all positive samples. Phylogenetic analysis and pairwise distance analysis indicated the highest homology to DWV type A (Figure 1).

**Figure 1.** Molecular phylogenetic analysis for RNA-dependent RNA polymerase of deformed wing virus (DWV) using the maximum likelihood method. The evolutionary history was inferred using the maximum likelihood method based on the Tamura–Nei model. The branch lengths of the tree measured the number of substitutions per site. The analysis involved 28 nucleotide sequences. There were 255 positions in the final dataset. Accession number, host, state, and year of available GenBank DWV sequences are shown. DWV sequence accession numbers are reported and associated with year and site of origin and type. The DWV sequence obtained from the tested *Aethina tumida* samples is in a red box.

> A similar analysis was conducted for the KBV sequence. A close relationship with sequences found in *A. mellifera* and *V. destructor* from the USA was detected (Figure 2).

**Figure 2.** Molecular phylogenetic analysis for RNA-dependent RNA polymerase of Kashmir Bee Virus (KBV) using the maximum likelihood method. The evolutionary history was inferred using the maximum likelihood method based on the Tamura–Nei model. The branch lengths of the tree measured the number of substitutions per site. The analysis involved 35 nucleotide sequences. There were 297 positions in the final dataset. Accession number, host, state, and year of available GenBank KBV sequences are shown. KBV sequence accession numbers are reported and associated with the year and site of origin. The DWV sequence obtained from the tested *Aethina tumida* samples is in a red box.
