## 4 Discussion  
Our data demonstrate the presence of SBPV-1 in Italian territory with a rather low level of positivity in most of the regions investigated, except for the Lazio and Emilia-Romagna regions, respectively, where positivity ranged from 56.40% to 59.46%. SBPV-1 was detected by molecular analysis in foraging bee samples and for seven of these the positivity was confirmed by Sanger sequencing of the amplification products. As reported, SBPV-1 shows higher prevalence and higher pathogenicity in bumblebees [7] than in honeybees; indeed, the Bombus species represents the ancestral host of this virus, whereas in _Apis mellifera_, the virus has subsequently adapted, maintaining a lower viral load than the primary species. This \"species-jump\" could have resulted in recombination events with loss of genomic portions within the same genome of SBPV-1 [2]. Maybe this can explain the presence of two different amplicons, 900 bp and/or 380 bp, was observed in our positive samples. The SBPV-1 lower positivity was already described by [PERSON] et al. (2010) [2]. They demonstrated through a study involving five European countries that the prevalence of SBPV was less than 2%, with positive samples found only in some colonies in England and Switzerland, with varying degrees of Varroa infestation. In our study, a further explanation of the low positivity of this virus is that the samples analyzed came from die-offs caused by the simultaneous presence of other viruses (ABPV, DWV). This reduces the survival rate of the colony, leading to rapid death and ultimately decreasing the possibility of detecting SBPV-1, meaning that this virus could be usually present as a subclinical infection with a low viral load and therefore is not detected by molecular analysis. Indeed, in literature [2], it is considered a rarely detected virus, and until 2010, it was associated with colony mortality only in Britain. The independence study between SBPV-1 and ABPV, as well as with DWV, did not show statistically significant associations among these viruses that share the same tropism for the nervous system [17]. This result is likely due to the small number of SBPV-1 and ABPV or SBPV-1 and DWV positive samples detected compared to the total number of samples. Although a possible association could be found between SBPV-1 and DWV-B, this should be confirmed by further studies employing random sampling. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Absence/Presence** & **DWV-B+** & **DWV-B\(-\)** & **TOT** \\ \hline SBPV-1+ & 69 & 1 & 70 \\ \hline SBPV-1\(-\) & 217 & 27 & 244 \\ \hline TOT & 286 & 28 & 327 \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Contingency table of SBPV-1 and DWV-B in foraging bee samples. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Absence/Presence** & **DWV+** & **DWV\(-\)** & **TOT** \\ \hline SBPV-1\(+\) & 67 & 19 & 86 \\ \hline SBPV-1\(-\) & 238 & 37 & 275 \\ \hline TOT & 305 & 56 & 361 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Contingency table of DWV and SBPV-1 in foraging bee samples. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Absence/Presence** & **ABPV+** & **ABPV\(-\)** & **TOT** \\ \hline DWV-B+ & 233 & 1 & 234 \\ \hline DWV-B\(-\) & 26 & 0 & 26 \\ \hline TOT & 259 & 1 & 260 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Contingency table of ABPV and DWV-B in foraging bee samples. For DWV-B, our data showed a strong presence of this virus in all Italian regions investigated, and no statistically significant association between DWV-B and ABPV viruses was found. It can be assumed that this result is due to the difficulty in obtaining samples quickly in cases of die-offs, which could lead to the degradation of the nucleic acid. The fact that DWV-B replicates very quickly in _Varroa destructor_[7; 18; 19] has probably contributed to the wide dissemination of this virus in the country [10], confirming the worldwide trends reported in the recent literature [10; 19]. Also, [PERSON] et al.