*4.2. RNA Extraction and RT-PCR Screening*

Each sample was homogenized with a sterile plastic pestle. Total RNA was extracted from the midgut tissues using TRIzol reagent (Invitrogen, Waltham, MA, USA) following the manufacturer's instructions. The quantity and purity of the RNA were measured using a ScanDrop2 Nanovolume spectrophotometer (Analytik Jena, Jena, Germany). For copy DNA (cDNA) synthesis, total RNA (1 μg) samples were treated with DNase I (Roche Molecular Biochemicals, Basel, Switzerland) and then primed with random hexamer primers and reverse-transcribed with Super Script III (Invitrogen, Waltham, MA, USA) at 42 ◦C for 3 h, after which the reaction was stopped at 70 ◦C.

All of the samples were first screened with the VP1-F/VP1-R specific primer set (Supplementary Table S2) via PCR with cycling at 95 ◦C initial denaturation for 45 s and then followed by 35 cycles of 95 ◦C denaturation for 45 s, 50 ◦C primer annealing for 45 s, and 72 ◦C extension for 1 min, followed by a 10 min final extension at 72 ◦C and storage at 20 ◦C. The RT-PCR products were analyzed by electrophoresis on a 2% agarose gel in 1× TAE buffer to check the SBV infection-positive samples for the following experiments. For the investigation of variations in VP1 region, the infection-positive samples were amplified by using VP1-F/SBV\_R4 primer set (Supplementary Table S2), and the PCR products were subjected to commercial DNA sequencing.

#### *4.3. Whole Genome Sequencing and Assembly of AcSBV and AmSBV*

The AcSBV infection-positive samples from *A. cerana* in Taipei City and the AmSBV infection-positive samples from *A. mellifera* in Yilan City were subjected to whole-genome sequencing by RT-PCR with 15 primer sets (Supplementary Table S2). PCR amplification was performed as described above. The RT-PCR products were analyzed by electrophoresis on a 2% agarose gel in 1× TAE buffer [35]. The PCR products with positive signals were purified (Geneaid, New Taipei City, Taiwan) and subjected to commercial DNA sequencing. The obtained sequences were subjected to the genome assembly using SeqMan (DNASTAR, Madison, WI, USA).

#### *4.4. Viral Genomic 5 and 3*-*End Sequencing*

The 5 and 3 untranslated regions of the AcSBV and AmSBV genomes were obtained by the rapid amplification of cDNA ends (RACE) method, which was slightly modified from [50]. For the 3 end of the viral genome, 1 μL of an anchor-dTv primer at 50 μM was used to prime 1 μg of total RNA in a 20 μL reaction containing 10 mM dNTPs at 70 ◦C for 5 min, after which the reaction mixture was placed on ice for 1 min. RNA was reverse transcribed by using Super-Script III (Invitrogen, Waltham, MA, USA) at 42 ◦C for 1 h, and the reaction was stopped by heating at 70 ◦C for 15 min. The viral 3 end sequences were amplified with genome-specific forward primers (GSP-F) and an anchor primer (Supplementary Table S2) using PCR Master Mix (Thermal, Riverside County, CA, USA).

The sequence of the viral 5 end was decoded as described by [51], with slight modifications [51]. A total of 5 μg RNA was used for 5- RACE, and the RN was primed with 0.5 μL of the GSP-RT primer (100 ng/μL) in a 20 μL reaction at 80 ◦C for 3 min, after which the mixture was rapidly transferred to ice. The iScript™ cDNA Synthesis Kit (BIO-RAD, Hercules, CA, USA) was used for reverse transcription at 42 ◦C for 1 h, and the reaction was inactivated at 95 ◦C for 5 min. Then, the RNA templates of the cDNA samples were digested with 1.5 U of RNase H (Thermo Fisher Scientific, Waltham, MA, USA) at 37 ◦C for 20 min. The sample was subsequently cleaned using a GenepHlow™ Gel/PCR Kit (Geneaid, New Taipei City, Taiwan). The 15 μL eluted cDNA sample was treated with transferase (Tdt) in the following reaction mixture: 5 μL of 10 × terminal deoxynucleotidyl transferase (Tdt) buffer (NEB, Ipswich, MA, USA), 5 μL CoCl2 (2.5 mM), 0.5 μL dATP (10 mM), and 0.5 μL Tdt for 5 end tailing, performed at 37 ◦C for 25 min, and the reaction was then stopped by heating at 70 ◦C for 10 min. The sample was next subjected to two rounds of PCR amplification using PCR Master Mix (Thermal, Riverside County, CA, USA). For the first round of PCR, 1 μL of cDNA template was used for amplification by three primers: GSP-R1, QO, and QT, at 25 pmols each (Supplementary Table S2). The PCR program was as follows: 98 ◦C initial denaturation for 5 min, 48 ◦C annealing for 2 min, 72 ◦C extension for 40 min, followed by 30 cycles of 94 ◦C denaturation for 10 s, 50 ◦C primer annealing for 30 s, 72 ◦C extension for 2 min, and a final extension at 72 ◦C for 15 min. The first-round PCR product was diluted 20-fold in ddH2O for the second round of amplification. A total of 1 μL of the diluted PCR product and 25 pmols of each of the

GSP-R2 and QI primers (Supplementary Table S2) were mixed for PCR amplification via the following program: 98 ◦C for 5 min, 30 cycles at 94 ◦C for 10 s, 50 ◦C for 30 s, 72 ◦C for 1 min, and a final extension at 72 ◦C for 15 min. The amplified PCR products were checked by electrophoresis on a 4% agarose gel in 1× TAE buffer. The amplified DNA fragments were purified using a GenepHlow™ Gel/PCR Kit (Geneaid, New Taipei City, Taiwan) and cloned into the TA vector (RBC Bioscience, New Taipei City, Taiwan); the ligated plasmid DNAs were transformed into *Escherichia coli* DH5α (RBC Bioscience, New Taipei City, Taiwan) following the user manual. The plasmids were extracted from cultured bacterial colonies with a Presto™ Mini Plasmid Kit (Geneaid, New Taipei City, Taiwan) and were sequenced bidirectionally with the M13F and M13R primers (Supplementary Table S2).

#### *4.5. Nucleotide Sequence Analysis and Comparison*

The genomes of AcSBV-TW and AmSBV-TW were annotated by using NCBI ORFfinder, and proteins were predicted by using NCBI BLASTp [51]. The nucleotide sequences and the amino acid sequences of these two viruses were further compared to each other or to those of other SBVs from other countries.

For the nucleotide sequences, the whole genome sequence, 5- UTR, ORF region, and 3- UTR were compared; for the amino acid sequences, the polyproteins, structural proteins (rhv\_like\_1 and rhv\_like\_2), and nonstructural proteins (helicase, protease, and RNAdependent RNA polymerase) of AcSBV-TW and AmSBV-TW were compared with other SBV sequence data from NCBI databases [52] (Table 1). Besides, the partial VP1 sequence of 3 AmSBV and 4 AcSBV from Taiwan were further compared to those of AmSBV-TW, AcSBV-TW, and other 54 SBVs. Multiple alignments of the sequences were obtained using ClustalX and edited in GeneDoc.

### *4.6. Phylogenetic Analysis*

Phylogenetic analysis was performed based on the polyprotein sequences and partial VP1 region of the SBVs as follows. For the polyprotein phylogenetic analysis, the sequences of 54 SBV strains were obtained from the GenBank database and aligned, compared with AcSBV-TW and AmSBV-TW by using ClustalX and GeneDoc. For the partial VP1 phylogenetic analysis, 3 AmSBV and 4 AcSBV from Taiwan were included. Molecular Evolutionary Genetics Analysis Version 7.0 (MEGA7) was used for phylogenetic analyses of these two conserved domains with the neighbor-joining method. The nodes were determined via bootstrap analysis with 1000 replicates [53].

#### **5. Conclusions**

The whole genomes of SBV strains from *A. mellifera* and *A. cerana* were determined, and the origin of AcSBV-TW was indicated to be close to China, while AmSBV-TW presented novel genomic features. The cross-infection of *A. mellifera* with AcSBV was demonstrated in the apiaries, where *A. mellifera* and *A. cerana* were crossbreeding in Northern Taiwan in our previous report [33], suggesting that the variations identified in the genomes of AcSBV-TW and AmSBV-TW. According to the whole genome data, the sequences of 5 and 3- UTR revealed divergence compared to the polyprotein coding sequences either between AcSBV-TW and AmSBV-TW or among those of SBV from other countries, assuming there is less evolutionary pressure on the untranslated regions of the viral genomes. A comparison of partial VP1 region in Taiwan SBVs and phylogenetic analysis showed a deletion feature in VP1 region. The deletion feature in VP1 region, also mainly observed in most of AcSBV in other regions, suggested the host-preference phenomenon. However, it should be noted that some AmSBV also have a deletion in the VP1 region, it might be a consequence of cross-infection and viral–host adaptions. Therefore, cross-infection might be a high-risk factor for SBV resurgence [18,30,37,40]. For long-term surveillance, the features of VP1 in the genome sequences of SBV strains might provide molecular markers for the detection of SBV adaption in different honeybee hosts. More detailed investigations of this issue will be needed in the future.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2076-0 817/10/1/14/s1, Supplementary Table S1: Information of sampling sites; Supplementary Table S2: Primers used in this study.

**Author Contributions:** Conceptualization, Y.-W.C. and Y.-S.N.; methodology, J.-C.C. and Y.-S.N.; validation, J.-C.C. and Y.-S.N.; formal analysis, J.-C.C., Z.-T.C. and C.-Y.K.; supervision, Y.-W.C. and Y.-S.N.; project administration, Y.-W.C. and Y.-S.N.; funding acquisition, Y.-W.C. and Y.-S.N.; writing—review and editing, J.-C.C., Z.-T.C., C.-Y.K., Y.-W.C. and Y.-S.N.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Ministry of Science and Technology, Taiwan, Grant MOST 107-2313-B-197-004-MY3 and 109-2313-B-005-048-MY3.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study, due to there is no any animal experiment involved the ethical issue.

**Data Availability Statement:** The sequences generated in this study were submitted to NCBI Gen-Bank and also are available from the corresponding author (Yu-Shin Nai) on reasonable request.

**Acknowledgments:** This research was supported by the Bureau of Animal and Plant Health Inspection and Quarantine, the Council of Agriculture and the Grant MOST 107-2313-B-197-004-MY3 from the Ministry of Science and Technology, Taiwan.

**Conflicts of Interest:** The authors declare there is no conflict of interest involved in this work.
