## 3 Results  
For the IZS LT 07/13 RC samples, 86 of the 361 foraging bee samples analyzed were positive for SBPV-1 (23.8%); 286 of the 307 samples tested positive for DWV-B (91.08%); 295 samples out of 322 were found positive for ABPV (93.95%), 31/314 for CBPV (9.87%) and 258/322 positive for DWV (82.17%).  
Emilia-Romagna (22/37 samples) and Lazio (22/39 samples) are the Italian regions that showed the highest presence of SBPV-1 in foraging bees, while in the Lombardia Region, only 2 positive samples were found out of the 37 tested (Figure 1).  
Regarding the SBPV-1, 33 samples showed a PCR product of 380 bp, 27 samples of 900 bp, and in 33 samples, both amplicons were detected. Sequencing analysis was possible only for those samples that showed a strong signal when visualizing by capillary electrophoresis. Four positive samples from Lazio had a 900 bp PCR product, as well as three 380 bp positive samples from Emilia-Romagna were confirmed by Sanger sequencing, and the sequences showed a percentage of identity greater than 98% with the sequence of Slow Bee Paralysis Virus strain PP, accession number KY243931, isolated in United Kingdom.  
Regarding DWV-B, all the samples from the Lazio (39) and Calabria (75) regions were found positive, while in the other regions, the percentage of positive samples was above 70% (Figure 1).  
DWV was detected in all Italian regions investigated, and it was present in all samples from the Lazio (39) and Campania (58) regions. In the remaining regions, the percentage of positive samples was between 56.8% in Emilia-Romagna and 93.5% in Trentino (Figure 1).  
ABPV was detected in all regions and was present in all samples from Trentino (46) and Emilia-Romagna (37), while in the remaining regions, the percentage of positive samples was between 43.1% in Campania and 97.3% in Lombardia (Figure 1).  
Compared to the other viruses, the level of positivity of CBPV was very low and was not detected in Emilia-Romagna, whereas in the other regions, it was between 2.13% (Toscana) and 21.65% (Calabria).  
In addition, in the voluntary bee health monitoring study (IZS LT 04/19 RC), only 1 sample (foraging bee) from the Lazio region of the 123 analyzed was positive for SBPV (0.81%), also confirmed by Sanger sequencing.  
For DWV-B, 18 of the 32 samples analyzed were positive (56.25%): 7 from Lazio (only foraging bees), 1 from Toscana, and 10 (only hive debris) from Emilia-Romagna, respectively.  
The presence of a possible association between SBPV-1 and DWV-B was evaluated in samples collected within the IZS LT07/13 RC. The positivity levels of these viruses were also compared with those of ABPV and DWV and with ABPV, respectively.  
The ABPV and DWV viruses were chosen for the independence study as they exhibit marked tropism for the nervous system and are not necessarily found in association [17]. No statistical evidence of association between the viruses was found (all \(p\) values > 0.05) (Tables 3-5). The independence study between SBPV-1 and DWV-B showed that there is a possible association between these two viruses given a \(p\) value of 0.0085 (Table 6).  
Figure 1: Bar graph showing the percentage of ABPV, CBPV, DWV, DWV-B, and SBPV-1 positive samples distributed in the various Italian regions with the number of positive samples over the total analyzed.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Absence/Presence** & **ABPV+** & **ABPV\(-\)** & **TOT** \\ \hline SBPV-1+ & 69 & 1 & 70 \\ \hline SBPV-1\(-\) & 245 & 7 & 252 \\ \hline TOT & 314 & 8 & 322 \\ \hline \hline \end{tabular}
\end{table}
Table 3: Contingency table of ABPV and SBPV-1 in foraging bee samples.