## 2 Materials and Methods
### Viral Investigations
#### 2.5.2 Molecular Analyses
The total number of individuals collected, those extracted for molecular analyses, their total weight, and the amount of diluent (physiological saline) used are reported. The remaining individuals were used for nsEM examination. ABPV/CBPV/DBVV viruses: A one-step Real-Time RT-qPCR was performed using 20 \(\upmu\)L of Master mix of QuantiFast(r) Pathogen RT-PCR + IC (QIAGEN) and 5 \(\upmu\)L of RNA, according to the listed thermal cycling conditions: 30 min at 50 \({}^{\circ}\)C (reverse transcription), 15 min at 95 \({}^{\circ}\)C (initial denaturation) followed by 45 denaturation cycles at 95 \({}^{\circ}\)C for 15 s and annealing/extension at 60 \({}^{\circ}\)C for 1 min for ABPV and CBPV; while for DWV the reverse transcription lasted only 20 min and the annealing/extension 70 s. Ten-fold serial dilution of cRNA transcripts, produced as described in [PERSON] et al. (2017) [61], (10\({}^{6}\)-10\({}^{2}\) viral RNA copies/\(\upmu\)L) were used to generate a standard curve and to quantify the RNA in the samples. Threshold cycles from our samples were compared with the standard curve, establishing a linear relation. The results were expressed in viral genome copies per individual using the following equation: copies/individual = 80 \(\times\) copies and (80/1.5) \(\times\) copies. This conversion factor was calculated based on the volume used at each step of the real-time RT-qPCR method. SBV/BQCV viruses: The one-step RT-PCR Kit (QIAGEN, Hilden, Germany) was used, with 22.5 \(\upmu\)L of Master Mix and 2.5 \(\upmu\)L of RNA, according to the listed thermal cycling conditions. The reverse transcription took place at 50 \({}^{\circ}\)C for 30 min and was followed by a denaturation and polymerase activation step at 95 \({}^{\circ}\)C for 15 min, and then 40 cycles of PCR, each consisting of 30 s at 94 \({}^{\circ}\)C, 50 s at 55 \({}^{\circ}\)C, and 1 min at 72 \({}^{\circ}\)C. Reactions were completed by a final elongation step at 72 \({}^{\circ}\)C for 7 min. The PCR products were electrophoresed in a 2.5% Tris-acetate-EDTAA-agarose gel and stained with GelRed Nucleic Acid Stain (BIOTIUM, Fremont, CA, USA). UV transillumination was used to visualize the \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Target** & **Forward Primer** & **Reverse Primer** & **Probe** & **Length** & **Type of** & **PCR** & **Reference** \\  & & & & & **Amplicon** & **Performed** & **Reverse** \\  & & & & (6-FAM) & & & \\ ABPV & GCCCAGACAAGCG & AGCCGGAAAACGC & TCCCC- & & & Real-Time & \\  & CAGTACT & GTCTT & GATAGCRAC & & & RT-PCR & \\  & & & & (6-FAM) & & & \\ CBPV & CGCAARTACGCCTT & ACTACTAGAACTC & TCAGAACCGAC & & & \\  & GATAAAGAAC & GTGCCTTCG & VACCCGCAAGTTC & & & RT-PCR & \\  & & & (BHQ-1) & & & \\  & & & (6 FAM) & & & \\ DWV & ATGGGTTTGATTCG/ & GATGTTCCG/AGGTTT & & & & \\  & AATACTTGGAAA & GGCTTTGATC & & & & RT-PCR & \\  & & & (MGB) & & & \\ SBV & ACCAACCGATTCCT & CCTTGGAAACTCTG & & & & end-point & \\  & CAGTAG & CTGTGTGTA & // & 487 & & & RT-PCR & \\ BQCV & AGTAGTTGATG & CTTAGCTTACTCG & & & & end-point & \\  & TACTTCC & CCACTT & // & 472 & & & RT-PCR & [63] \\ IAPV & GAGCTGCGATCC & TCCATTACCACT & & & & end-point & \\  & & & & & & RT-PCR & \\ KBV & GATGAACGTGCAC & TGTGGTGCTGCTA & & & & end-point & \\  & & & & & RT-PCR & \\  & & & & & & RT-PCR & \\ _Nosema_ & CCAGATGGTGATA & CACATTATTACT & & & & end-point & \\ _apis_ & TGAGATG & TCAGATCATA & & & & RT-PCR & \\ _Nosema_ & CGGCGACGATGGTG & CCCGGTCATTCTC & & & end-point & \\ _cernane_ & ATTAGAAAATATTAA & AAACAAAAACCCG & & & RT-PCR & [66] \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Primers and Taqman probes used for RT-PCR/PCR real-time and RT-PCR/PCR end-point assays [ABPV = Acute Bee Paralysis Virus; CBPV = Chronic Bee Paralysis Virus; DWV = Deformed Wing Virus; BQCV = Black Queen Cell Virus; SBV = Sac Brood Virus; KBV = Kashmir Bee Virus; IAPV = Israeli Acute Paralysis Virus].