*2.3. Phylogenetic Analysis*

Phylogenetic analysis was performed based on the polyprotein sequences of 56 strains of SBV. The phylogenetic tree clearly diverged into two main branches according to the host (Figure 2). The first branch was composed of the SBV strains from *A. mellifera*; within this branch, AmSBV-TW was closely related to AmSBV-UK and AmSBV-Kor19. The second branch was composed of SBV strains from either *A. cerana* or *A. mellifera*; moreover, a branch contained two groups, one composed of AcSBV from India, while the other consisted of AcSBV and AmSBV from Asian areas, which included AcSBV-TW from Taiwan, CSBV strains from China, AmSBV/AcSBV from Korea, and AmSBV/AcSBV from Vietnam. Especially according to the phylogenetic tree, AcSBV-TW is closely related to AmCSBV-SDLY and CSBV-FZ (Figure 2).

### *2.4. Variation of VP1 Region in AcSBV and AmSBV in Taiwan*

As aforementioned, the deletion of 51 base pairs (17 amino acids) in the VP1 region were found in most of AcSBV and, thereby, named as AC-genotype and vice versa (AMgenotype without any deletion in the VP1 region). To better understand whether the AcSBV AM-genotype and AmSBV AC-genotype exist in the populations of *A. cerana* and *A. mellifera,* the partial VP1 sequence of three AmSBV and four AcSBV from Taiwan were further compared to those of AmSBV-TWand AcSBV-TW (Figure 3A). The results showed that the 17-amino-acid deletion (AC genotype) was only detected in one AmSBV sample in Taichung; besides, one AcSBV with AM genotype was also detected in the sample from Hsinchu (Figure 3A) The phylogenetic analysis was also performed based on the partial VP1 region of 63 strains of SBV. It revealed similar result to those of polyprotein phylogeny. Moreover, the AcSBV-AC genotype and AmSBV-AC genotype in Taiwan were grouped in the same clade, which was closed to CSBV-FZ and CSBV-JL, and the AmSBV-AM genotype and AcSBV-AM genotype in Taiwan were grouped in the same clade, which was closed to AmSBV-UK (Figure 3B). These results supported that the cross-infection between AcSBV and AmSBV in *A. cerana* and *A. mellifera.*

**Figure 3.** Comparison and phylogenetic analysis of partial VP1 region of AcSBV and AmSBV from Taiwan. (**A**) The pairwise alignment indicated the 17-anmion deletion presented not in AcSBV but also AmSBV vice versa. (**B**) Phylogenetic tree construct based on the partial VP-1 amino acid sequences of Taiwan AmSBV and AcSBV, and other 54 SBV strains from NCBI. The phylogenetic tree was constructed using the neighbor-joining (NJ) method and 1000 bootstrap replications. red font: The Taiwan sequences from this study. \*: A note for every 10 bases.

#### **3. Discussion**

AcSBV has recently been recorded in Taiwan and caused serious losses of *A. cerana* from 2015 to 2019 [33–35]. In Taiwan, most *A. cerana* populations are reared in Northern Taiwan, and according to our observations, some of these apiaries are crossbreeding with *A. mellifera* populations. The detection of AcSBV prevalence in *A. mellifera* populations from the sampling sites where *A. cerana* and *A. mellifera* were crossbreeding confirmed that AcSBV prevalence rates gradually developed a similar trend in the *A. cerana* and *A. mellifera* crossbreeding apiaries, and the existence of AcSBV cross-infection between *A. cerana* and *A. mellifera* was also confirmed by phylogenetic analysis based on partial VP1 sequences [33]. Similar to our case, some SBV strains from *A. mellifera* included in this study were found to be distinct from other AmSBV strains in terms of genomic features and were clustered with AcSBVs based on whole genome comparisons and phylogenetic analysis [24,30,36,40]. Strains from the same or closer geography distance showed higher similarity, and the phylogenetic analysis also indicated the same result [24,30,36,40,44]. Indeed, it was shown that the cross-infection of SBV strains occurs between two honeybee species in other countries, including China, Vietnam, and Korea, leading to the high genetic divergence among SBV strains [24,30,36,40].

As mentioned above, the comparison of different genome sequences could provide precise and reliable information for detecting variations within closely related species. In this study, complete SBV genome sequences from *A. cerana* and *A. mellifera* in Taiwan were determined and were designated AcSBV-TW and AmSBV-TW, respectively. Our comparisons revealed greater divergence in 5 and 3- UTRs than in ORF region not only between AmSBV-TW and AcSBV-TW but also compared with those of SBVs from other countries (Table 3). The structures of 5- UTR play many functions in RNA viruses, including viral replication, translation, virus–host protein interactions, and virulence [45,46]. It has been also reported that the 5- UTR of RNA viruses in *Iflaviridae* functions as an internal ribosome entry site (IRES) [47]. Similar result was also described from [30], that 5- UTRs of VN-SBVs (including AcSBV and AmSBV in Vietnam) showed greater divergence from SBV strains from other countries [30]. The structure of 5- UTR of SBV might have a critical function for virus replication, therefore the sequence divergence may reveal different viral activities among different viruses.

It should be noted that deletions in the 712–730 amino acid (VP1) region of the ORF were found in most AcSBVs [24,30,40]. Since it has been mentioned that VP1 has the highest sequence variation among SBVs [24,30,40,48]. Based on our comparisons, there are three types of deletion patterns: 17-amino-acid deletions, 10–13-amino-acid deletions, and deletions of less than 10 amino acids (Figure 2). Most of the examined AcSBVs, including AcSBV-TW, exhibit a 17- or 10–13-amino-acid deletion in VP1 region, while there were nine AcSBVs from China, India, and Vietnam exhibiting no deletions, and a deletion of less than 10 amino acids was found in SBVs from *A. mellifera* in Australia, including AmSBV-VN3 and SA. AcSBVs from India all have 10-amino-acid deletion, which were clustered in same branch, suggesting that the occurrence of the 17-amino-acid difference in the VP1 region tends to be host-preference. Interestingly, some AmSBV from Asia countries, where have *A. cerana* population, including AmSBV-Viet4 and AmSBV-Kor19, also harbor the same 17-amino-acid deletion in their VP1 region, indicating the cross-infection of SBV at different geographic origins [49].

Further investigation of the VP1 variations of AcSBV and AmSBV in Taiwan indicated that most AcSBVs have 17-amino-acid deletion in their VP1 region compared to AmSBV, while the AmSBV-AC genotype and AcSBV-AM genotype were also detectable in AmSBV and AcSBV, respectively. It has been reported that high variability exists among SBV genomes, especially between AC-genotype SBV and AM-genotype SBV, and this genetic diversity is supported by the geographic distances or viral cross-infections between different honeybee species [30,40]. These characteristics might also provide clues regarding SBV adaption in different hosts [24,30]. In conclusion, the genomic differences in AmSBV-TW and AcSBV-TW compared with other SBVs could be further applied to identify genetic markers for host-specific and geographic distance evaluations.

The phylogenetic analysis based on the polyproteins and partial VP1 region of AmSBV-TW and AcSBV-TW and other SBV strains revealed that the SBV strains diverged into two distinct branches, which could represent host affiliation and geographic origin. According to comparisons with the current 54 strains of SBV available in NCBI, AmSBV-TW and AcSBV-TW were grouped onto different branches. AcSBV-TW is closely related to AmCSBV-SDLY and CSBV-FZ and was clustered into the AcSBV group with the AC genotype; therefore, it was assumed that the AcSBV in Taiwan may have originated from China and currently be experiencing host adaption and evolution. In contrast, AmSBV-TW was grouped into the AM-genotype SBVs, which originated from *A. mellifera*; however, AmSBV- TW was separated from other AmSBVs in this group, suggesting that geographic distance might be involved in the process of genomic divergence.

The comparison and phylogenetic analysis of partial VP1 region in another seven SBVs in Taiwan showed that most of AcSBV and AmSBV were grouped into AcSBV-TW and AmSBV-TW, respectively, except one AmSBV-AC genotype (grouped with the AcSBV-TW) and one AcSBV-AM genotype (grouped with the AmSBV-TW). These results suggested that AcSBVs in Taiwan presented the closely geographical relationship to those of China, while AmSBVs in Taiwan revealed the geographic distance-based evolution. Additionally, the AcSBV-AM genotype and AmSBV-AC genotype clearly showed the viral cross-infection between these two species.
