*4.2. RNA Extraction, Reverse Transcription (RT) and PCR*

A total of 15/40 honeybees were subjected to biomolecular investigation to verify and, in case of positive results, quantify the presence of viral RNA.

Samples were individually chopped up with a sterile blade to facilitate subsequent homogenization with the TissueLyser mechanical homogenizer (Qiagen, Hilden, Germany). Each sample was put in 2 mL tubes along with a grinding metal bead and subjected to lysis by two steps of five minutes at 50 Hz, interspersed with a cycle of ice cooling of 2 min to avoid overheating and preserve the integrity of the biological molecules.

RNA was extracted and purified from genomic DNA using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), according to the protocol provided by the manufacturer, and RNA concentration was measured by spectrophotometric reading.

For each sample, 250 ng of RNA were subjected to RT using the commercial iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's recommendations.

Subsequently, 12.5 ng of cDNA for each sample were subjected to PCR to amplify a segment of DWV genetic material and verify the presence/absence of the virus in the samples using the AmpliTaq Gold DNA Polymerase kit (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA) according to manufacturer's instructions. The housekeeping gene β-actin (Act β) of A. mellifera was also amplified to ensure the presence of amplifiable cDNA in each sample. One no template control (NTC) was included in each PCR reaction as negative control.

Subsequently, a new PCR was performed on the same samples to discriminate between the two different variants DWV-A and DWV-B according to the protocols found in the literature [64,65].

Moreover, a multiplex PCR was executed on the previous samples (15/40) to screen for the presence of six other relevant honeybee viruses (ABPV, CBPV, SBV, BQCV, KBV, IAPV) according to the protocol proposed and validated by Cagirgan and Yazici [66]. The set of primers used for amplification of the genetic material of viruses and Act β used in this study were found in literature and a complete list, together with the product size, annealing temperature and application is reported in Supplementary Materials.

Amplification products were migrated by electrophoresis on 2.5% agarose gel in TBE buffer (Tris-Borate-EDTA) along with a 50 bp molecular marker (Bioline), stained with ethidium bromide and observed under UV with the ChemiDoc gel scanner (Bio-Rad).

#### *4.3. Real-Time PCR (qPCR) for Detection of Relative Viral Load*

In order to determine a relative quantization (RQ) of viral load of the samples, a Real-Time PCR (qPCR) was carried out using the primers described above.

For each sample tested positive for DWV in PCR, 12.5 ng of cDNA were subjected to qPCR using iTaq Universal SYBR Green Supermix kit (Bio-Rad), according to the manufacturer's instructions.

Amplification of honeybee Act β as reference gene was also performed in parallel to allow normalization of the results and an NTC was included in the reaction as negative control.

Relative quantization of DWV viral load was calculated by using the 2−ΔΔCq method as previously described [67,68]. Briefly, fold change in viral load was estimated for each individual S sample against A samples considered as control group.

#### *4.4. Histopathological Analysis*

Samples were processed as previously described [69]. Briefly, honeybees were individually injected with 10 μL of 10% buffered formalin and then stored for 24 h in 50 mL tubes containing the same fixative.

Subsequently, each sample was placed in an embedding cassette and processed. 3 μm sections were cut, stained with hematoxylin and eosin, and observed by light microscopy (Microscope Nikon Eclipse E-600, Tokyo, Japan). All tissues were observed to identify possible alterations and analyzed for the presence of visible pathogens, i.e., Nosema spp.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/pathogens10070874/s1, File S1: Oligonucleotides used for amplification of viruses and Act β in this study. Sequences, products size, annealing temperature and applications are indicated.

**Author Contributions:** Conceptualization, K.P. and P.M.; Methodology, K.P. and G.A.; Validation, M.M., N.P. and P.M.; Formal Analysis, N.P.; Investigation, K.P. and G.A.; Writing—Original Draft Preparation, K.P.; Writing—Review and Editing, M.M. and P.M.; Supervision, P.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by PSR 14/20 Campania. Tipologia intervento 16.1.1 "sostegno per costituzione e funzionamento dei GO del PEI in materia di produttività e sostenibilità dell'agricoltura". Azione 2 "Sostegno ai POI". "Uso tecnologico e muove pratiche a carattere innovativo per la gestione, il controllo e la valorizzazione economica del cinghiale (Sus scrofa) in maniera sostenibile in Regione Campania". S.U.S Campania (CUP B58H19004460009).

**Institutional Review Board Statement:** Ethical review and approval were waived for this study, as according to the D.L. 4 March 2014 n.26, and national implementing decree following the European regulation 2010/63/UE, ethical approval is not necessary for insects with the except of cephalopoda. **Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are available on reasonable request to the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.
