**1. Introduction**

Sacbrood virus (SBV) is a single-stranded, positive-sense RNA virus that belongs to the *Iflaviridae* family [1–3]. The particles of SBV are 28 nm in diameter, nonenveloped, icosahedral [4,5]. SBV is a common honeybee virus that exhibits a high prevalence of infection mainly in early larval stage of honeybees. This condition affects the broods of honeybees, and the specific symptoms can be easily identified in dead deformed larvae in hives with fluid-filled sacs [6–9].

Sacbrood disease, which is caused by SBV infection, was first reported and verified in *Apis mellifera* in 1964 [7,10]. The ectoparasitic mite *V. destructor* plays a role in SBV transmission [11]. Infection with SBV (AmSBV) is now commonly found in *A. mellifera* worldwide and does not usually result in *A. mellifera* colony loss [12–16]. However, according to a previous report from 1976, the SBV found in *A. cerana* (AcSBV) has large impacts on *A. cerana* in several Asian countries, including China, Korea, India, Vietnam, and Thailand [17–22]. During 1991–1992, an outbreak of sacbrood disease caused up to 90% colony losses in Thailand [23–30]. As mentioned above, AcSBV infection usually causes a high rate of *A. cerana* larvae death and may even lead to whole-colony collapse [31,32].

The *A. cerana* is an indigenous honeybee species in Taiwan. The natural fitness of *A. cerana* is better than *A. mellifera*. The *A. cerana* has a higher tolerance for low tempera-

**Citation:** Chang, J.-C.; Chang, Z.-T.; Ko, C.-Y.; Chen, Y.-W.; Nai, Y.-S. Genomic Sequencing and Comparison of Sacbrood Viruses from *Apis cerana* and *Apis mellifera* in Taiwan. *Pathogens* **2021**, *10*, 14. https://dx.doi.org/10.3390/ pathogens10010014

Received: 1 December 2020 Accepted: 22 December 2020 Published: 28 December 2020

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ture and better performance on the pollination in the mountain regions than *A. mellifera*. Therefore, *A. cerana* contributes to the pollination of mountainous orchards, including plums and peaches, etc., which are counted for 1.3% of Taiwan agricultural production [33]. Since AcSBV was first detected in Taiwan in 2015, many beekeepers in Taiwan have reported significant *A. cerana* larval death with symptoms caused by AcSBV in *A. cerana* colonies [34,35]. The virus was found to have spread from southern Taiwan to northern and then eastern Taiwan in 2016. Based on long-term surveillance data, the prevalence rate of AcSBV in *A. cerana* colonies had dramatically increased from 47% to ~70% at the end of 2016 and continued to increase to 72% in 2017 [35]. More than 90% of *A. cerana* colonies were influenced by AcSBV infection from 2016–2019, and the colony collapse does have impacts on the sale market of *A. cerana* colonies, price of honey, and the productions of mountainous orchards. The prevalence of SBV in the population of *A. cerana* in Taiwan is now irreversible.

To better understand the relationship between each SBV strain among Asian countries, the analysis of genome sequences from different geographic areas could provide an accurate and reliable method of detecting variations within the same type of genome based on molecular comparisons. Several studies have examined the whole genome sequences of either AmSBV or AcSBV worldwide. In Korea, six AmSBVs (AmSBV-Kor1 [KP296800.1], AmSBV-Kor2 [KP296801.1], AcSBV-Kor3 [KP296802.1], AcSBV-Kor4 [KP296803.1], AmSBV-Kor19 [JQ390592.1], and AmSBV-Kor21 [JQ390591.1]) from *A. mellifera* were sequenced and further compared (Table 1) [24,36]. In 2017, a comparative genomic analysis among nine SBVs of *A. cerana* and *A. mellifera* was performed in Vietnam [30]. These reports identified different genomic features and revealed the genetic diversity among these SBVs, suggesting that viral cross-infections might occur between AcSBV and AmSBV.

**Table 1.** Information on the sacbrood virus (SBV) strains used in this study.



**Table 1.** *Cont.*


According to our previous data, cross-infection might occur between AcSBV and AmSBV in Taiwan [33,35]. However, the available information on the whole genome sequences of AcSBV and AmSBV in Taiwan is insufficient. Therefore, this study attempted to determine and analyze two complete genome sequences of AcSBV and AmSBV in Taiwan. This is the first complete genome sequences of AcSBV and AmSBV from Taiwan. Phylogenetic analysis based on conserved viral proteins and similarity comparisons of the genomic sequences with those of 54 other SBV strains worldwide were also performed, these results may contribute to better understanding the variation of other SBV strains.

#### **2. Results**

#### *2.1. Genomic Sequences and Analysis of SBV Strains in Taiwan*

The whole genomes of AmSBV and AcSBV from *A. mellifera* and *A. cerana* in Taiwan were sequenced. The complete genome sequences of the two SBV strains were deposited in GenBank under the accession numbers MN082651 for AmSBV-TW and MN082652 for AcSBV-TW. The genomes of AmSBV-TW and AcSBV-TW were annotated by using NCBI ORF finder, and the numbers of RNAs encoded by AmSBV-TW and AcSBV-TW were 8885 and 8776, respectively. The 5 and 3 untranslated regions (UTRs) of AmSBV-TW were 212 and 115 nt, respectively; for AcSBV-TW, the 5 and 3- UTRs were 174 and 272 nt, respectively. Only one open reading frame (ORF) was predicted in the genomic RNA sequence of AmSBV-TW, which extended from nt 213 to 8792, encoding a putative polyprotein of 2859 amino acids. The genomic RNA of AcSBV-TW also encoded one ORF, from nt 175 to nt 8700, encoding a putative polyprotein of 2841 amino acids (Figure 1). Two structural domains were identified as rhv-like domains in the 5 region of AmSBV-TW and AcSBV-TW, and three nonstructural domains, including *helicase*, *protease,* and *RNAdependent RNA polymerase* (*RdRp*), were located at the 3regions of both AmSBV-TW and

AcSBV-TW. The analysis of the protein domain arrangement and genomic structures of AmSBV-TW and AcSBV-TW revealed characteristics of family *Iflaviridae* (Figure 1).

**Figure 1.** The genomic maps of (**A**) AmSBV-TW (accession number: MN082651) and (**B**) AcSBV-TW (accession number: MN082652). The full-length sequences were obtained using a combination of RT-PCR amplification and rapid amplification of 5 and 3 cDNA ends (5- RACE and 3- RACE). The nucleotide (nt) and amino acid (aa) positions of each domain was indicated below the schematic of AmSBV-TW and AcSBV-TW, respectively. The 5 terminal sequences of AmSBV-TW and AcSBV-TW were determined by 5- RACE, and the prediction of the 5 secondary structure of AmSBV-TW and AcSBV-TW was performed on the RNAfold WebServer (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) and presented in the dotted box. VPg = viral protein genomic linked. \* Mismatch nucleotide base.

*2.2. Comparisons of SBV Strains*

The sequences of AmSBV-TW and AcSBV-TW were first compared to each other (Table 2). The results of nt sequence comparisons showed that the full-length genomic RNA and ORF regions were highly conserved between AmSBV-TW and AcSBV-TW, sharing 90% identity, while the 5 and 3- UTRs showed high variation between AmSBV-TW and AcSBV-TW, presenting 68% and 73% identity, respectively (Table 2). In the amino acid sequence comparisons, the helicase protein domain exhibited the highest identity (99%) and was the most conserved protein domain between AmSBV-TW and AcSBV-TW, followed by rhv\_like\_2 (97%), RdRp (96%), polyprotein, and rhv\_like\_1 (95%), while the nonstructural protein protease showed low identity (75%) between AmSBV-TW and AcSBV-TW (Table 2).


**Table 2.** Comparison of genomic sequences and protein regions of AcSBV and AmSBV in Taiwan.

The genomic regions of AmSBV-TW and AcSBV-TW were further compared to those of SBV strains from other countries (Table 3). The nucleotide sequences of the whole AmSBV-TW and AcSBV-TW genomes shared identities of 87% (AcSBV-India-II10) to 92% (AcSBV-Viet-SBM2) and 88% (South Australia\_1, 2, 3, SBV\_MR, MD1, 2 and AcSBV-India-II10) to 96% (AcSBV-Viet1, 2, AmCSBV-SDLY and CSBV-FZ), respectively, with other SBVs (Table 1; Table 3). The identities of the 5 and 3- UTRs showed high variation among SBVs; for AmSBV-TW, the 5 and 3- UTRs shared 30% (CSBV-SXnor1) to 78% (Korean strain) and 10% (AcSBV-India-K5B) to 94% (MD1 strain) identities, respectively, with those of other SBVs, while AcSBV-TW showed 42% (CSBV-SXnor1) to 93% (AcSBV-Viet3) identity for the 5- UTR and 11% (AcSBV-India-K5B) to 85% (NT strain) identity for the 3- UTR (Table 1; Table 3).

**Table 3.** Comparison of the nucleotide sequence homology (%) of different genomic regions of AcSBV-TW, AmSBV-TW, and 54 other SBV strains.



**Table 3.** *Cont.*


The amino acid identities among the SBV strains were similar to the variations in the nucleotide identities. In the comparison of polyprotein amino acid sequences, AmSBV-TW was most similar to SBV-UK, with 98% aa identity, and AcSBV-TW was most similar to AcSBV-Viet1 and 2, sharing 98% aa identity. (Table 4). Among the structural proteins (rhv\_like\_1 and rhv\_like\_2), AmSBV-TW shared 81% (AmSBV-Viet6) to 98% (NT strain) and 94% (AcSBV-Viet-NA) to 99% (AmSBV-Kor1) identities with those of other SBVs, and AcSBV-TW shared 81% (AmSBV-Viet6) to 98% (CSBV-FZ) and 94% (AcSBV-Viet-BP) to 100% (AmCSBV-SDLY, CSBV-JLCBS-2014 and AcSBV-Viet-BG) identities with those of other SBVs (Table 4). The identities of the nonstructural proteins, including helicase, protease and RdRp, between AmSBV-TW and other SBVs showed greater variation than those of the structural proteins, ranging from 60–100%, 70–98%, and 88–98%, respectively, while the corresponding values were 60–100%, 67–99%, and 89–98% for AcSBV-TW (Table 4).

*Pathogens* **2021**, *10*, 14

