## 2 Materials and Methods
### RT-PCR SBPV-1  
For SBPV-1 detection, the primers described by [PERSON] 2017 [14] (Primer SBPV-1 Fw: 5\({}^{\prime}\)-GATTTGCCGGAATCGTAATATTGTTTG-3\({}^{\prime}\); Primer SBPV-1 Rv: 5\({}^{\prime}\)-ACCAGTTAGTACA CTCCTGTAACTTG-3\({}^{\prime}\)), targeting a region of RNA-dependent RNA polymerase (RdRP), were used. PCR was performed using AmplTaq Gold\({}^{\otimes}\) DNA Polymerase kit (Applied Biosystems, Thermo Fisher Scientific). The master mix, in a final volume of 5 \(\upmu\)L, included 5 \(\upmu\)L cDNA and a final concentration of 1\(\times\) Buffer, 3 mM MgCl2, 0.4 \(\upmu\)M for each primer, 0.2 \(\upmu\)M dNTPs, and 3 U AmplTaq Gold\({}^{\otimes}\) DNA Polymerase. The amplification was carried out using a VerityTM 96-Well Thermal Cycler (Applied Biosystems, Thermo Fisher Scientific,  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Virus.** & \begin{tabular}{c} **TARGET** \\ **GENE** \\ \end{tabular} & **Primer and Probe** & **Sequence (5\({}^{\prime}\)-3\({}^{\prime}\))** & **Ref.** & \begin{tabular}{c} **Final Concentration** \\ **(\(\upmu\)M)** \\ \end{tabular} \\ \hline DWV-B & VP3 & \begin{tabular}{c} F-VDV1\_4218 \\ R-VDV1\_4290 \\ Probe VDVI\_4266 \\ \end{tabular} & \begin{tabular}{c} GGTCTCGAACCGAAAATAG \\ CTAGCATATCCATGATATAAAAC \\ \end{tabular} & [13] & \begin{tabular}{c} 1.2 \\ 1.2 \\ 0.4 \\ \end{tabular} \\ \hline DWV & gp1 & \begin{tabular}{c} DWV Forward \\ DWV Reverse \\ DWV Probe \\ \end{tabular} & \begin{tabular}{c} ATGGGTTTGATTGG /AATATCTTGGAA \\ CTGTGTCCG/AGGTGCTTTAATGA \\ \end{tabular} & [14] & \begin{tabular}{c} 0.9 \\ 0.9 \\ 0.9 \\ 0.25 \\ \end{tabular} \\ \hline ABPV & gp1 & \begin{tabular}{c} APV-1 Forward \\ APV-1 Reverse \\ ABPV-1 Probe \\ \end{tabular} & \begin{tabular}{c} GCCCGAAAGACCGCGTACT \\ APV-1 Reverse \\ ABPV-1 Probe \\ \end{tabular} & [14] & \begin{tabular}{c} 0.9 \\ 0.9 \\ 0.25 \\ \end{tabular} \\ \hline CBPV & RdRP ORF 3 & \begin{tabular}{c} CBPV Forward \\ CBPV Reverse \\ CBPV Probe \\ \end{tabular} & \
\begin{tabular}{c} CCCCAAGATGCCCTTGATAAAAGAC \\ CTACTAGAACACCTGCCCTTCG \\ \end{tabular} & [15] &
\begin{tabular}{c} 0.3 \\ 0.3 \\ 0.3 \\ 0.3 \\ 0.3 \\ 0.3 \\ 0.3 \\ 0.3 \\ \end{tabular} \\ \hline \hline \end{tabular}
\end{table}
Table 2: List of primers and probes used for virus detection (DWV-B, DWV, ABPV, CBPV).  
Waltham, MA, USA). The thermal cycling profile consisted of an initial step at 94 \({}^{\circ}\)C for 10 min, followed by 40 cycles consisting of denaturation at 95 \({}^{\circ}\)C for 1 min, annealing at 58 \({}^{\circ}\)C for 1 min, extension at 72 \({}^{\circ}\)C for 1 min, and a final elongation step at 72 \({}^{\circ}\)C for 10 min. Negative and positive controls were included in each PCR. The SBPV-1 PCR products (900 bp or 380 bp depending on the circulating strains) [2] were visualized by capillary electrophoresis on a QIAxcel advanced instrument (QIAGEN, Hilden, Germany) and some were confirmed by Sanger sequencing using the kit BigDye terminator Cycle Sequencing Reading Reaction Mix V 3.1 (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA USA). The nucleotide sequences obtained were analyzed using Basic Local Alignment, Search Tool (BLAST) by comparing them to sequences from reference strains of different SBPV virus present in the NCBI GenBank ([[http://www.ncbi.nlm.nih.gov/](http://www.ncbi.nlm.nih.gov/)]([http://www.ncbi.nlm.nih.gov/](http://www.ncbi.nlm.nih.gov/)), accessed on 22 September 2022).