Seung-Won Kang

Sacbrood virus (SBV) represents a serious threat to the health of managed honeybees. We determined four complete SBV genomic sequences (AmSBV-Kor1, AmSBV-Kor2, AcSBV-Kor3, and AcSBV-Kor4) isolated from Apis mellifera and Apis cerana in various regions of South Korea. A phylogenetic tree was constructed from the complete genomic sequences of these Korean SBVs (KSBVs) and 21 previously reported SBV sequences from other countries. Three KSBVs (not AmSBV-Kor1) clustered with previously reported Korean genomes, but separately from SBV genomes from other countries. The KSBVs shared 90–98 % identity, and 89–97 % identity with the genomes from other countries. AmSBV-Kor1 was least similar (~90 % identity) to the other KSBVs, and was most similar to previously reported strains AmSBV-Kor21 (97 %) and AmSBV-UK (93 %). Phylogenetic analysis of the partial VP1 region sequences indicated that SBVs clustered by host species and country of origin. The KSBVs were aligned with nine previously reported complete SBV genomes and compared. The KSBVs were most different from the other genomes at the end of the 5′ untranslated region and in the entire open reading frame. A SimPlot graph of the VP1 region confirmed its high variability, especially between the SBVs infecting A. mellifera and A. cerana. In this genomic region, SBVs from A. mellifera species contain an extra continuous 51-nucleotide sequence relative to the SBVs from A. cerana. This genomic diversity may reflect the adaptation of SBV to specific hosts, viral cross-infections, and the spatial distances separating the KSBVs from other SBVs.

Kashmir bee virus (KBV) is one of the most common viral infections in honeybees. In this study, a phylogenetic analysis was performed using nine partial nucleotide sequences of RdRp and the structural polyprotein regions of South Korean KBV genotypes, as well as nine previously reported KBV genotypes from various countries and two closely related genotypes of Israeli acute paralysis virus (IAPV) and Acute bee paralysis virus (ABPV). The Korean KBV genotypes were highly conserved with 94–99 % shared identity, but they also shared 88–95 % identity with genotypes from various countries, and they formed a separate KBV cluster in the phylogenetic tree. The complete genome sequence of Korean KBV was also determined and aligned with previously reported complete reference genome sequences of KBV, IAPV, and ABPV to compare different genomic regions. The complete Korean KBV genome shared 93, 79, and 71 % similarity with the complete reference genomes of KBV, IAPV, and ABPV, respectively. The Korean KBV was highly conserved relative to the reference KBV genomes in the intergenic and 3′ untranslated region (UTR), but it had a highly variable 5′ UTR, whereas there was little divergence in the helicase and 3C-protease of the nonstructural protein, and the external domains of the structural polyprotein region. Thus, genetic recombination and geographical distance may explain the genomic variations between the Korean and reference KBV genotypes.

Black queen cell virus (BQCV) infection is one of the most common viral infections in honeybees (Apis mellifera). A phylogenetic tree was constructed for 19 partial nucleotide sequences for the capsid region of South Korean BQCV, which were also compared with 10 previously reported BQCV sequences derived from different countries. The Korean BQCV genomes were highly conserved and showed 97–100 % identity. They also showed 92–99 % similarity with other country genotypes and showed no significant clustering in the phylogenetic tree. In order to investigate this phenomenon in more detail, the complete genome sequence of the Korean BQCV strain was determined and aligned with those from a South African reference strain and European genotypes, Poland4–6 and Hungary10. A phylogenetic tree was then constructed. The Korean BQCV strain showed a high level of similarity (92 %) with Hungary10, but low similarity (86 %) with the South African reference genotype. Comparison of the Korean and other sequences across different genome regions revealed that the 5′-UTR, the intergenic region, and the capsid regions of the BQCV genome were highly conserved. ORF1 (a non-structural protein coding region) was more variable than ORF2 (a structural protein coding region). The 5′-proximal third of ORF1 was particularly variable and contained several insertions/deletions. This phenomenon may be explained by intra-molecular recombination between the Korean and other BQCV genotypes; this appeared to have happened more with the South African reference strain than with the European genotypes.

The black queen cell virus (BQCV), a picorna-like honeybee virus, was first isolated from queen larvae and pupae of honeybees found dead in their cells. BQCV is the most common cause of death in queen larvae. Phylogenetic analysis of two Apis cerana and three Apis mellifera BQCV genotypes collected from honeybee colonies in different regions of South Korea, central European BQCV genotypes, and a South African BQCV reference genotype was performed on a partial helicase enzyme coding region (ORF1) and a partial structural polypeptide coding region (ORF2). The phylogeny based on the ORF2 region showed clustering of all the Korean genotypes corresponding to their geographic origin, with the exception of Korean Am str3 which showed more similarity to the central European and the South African reference genotype. However, the ORF1-based tree exhibited a different distribution of the Korean strains, in which A. cerana isolates formed one cluster and all A. mellifera isolates formed a separate cluster. The RT-PCR assay described in this study is a sensitive and reliable method for the detection and classification of BQCV strains from various regions of Korea. BQCV infection is present in both A. cerana and A. mellifera colonies. With this in mind, the present study examined the transmission of honeybee BQCV infections between A. cerana and A. mellifera.

Nuclear ribosomal DNA sequence of the second internal transcribed spacer (ITS-2) has been used efficiently to identify the liver fluke species collected from different hosts and various geographic regions. ITS-2 sequences of 19 Fasciola samples collected from Korean native cattle were determined and compared. Sequence comparison including ITS-2 sequences of isolates from this study and reference sequences from Fasciola hepatica and Fasciola gigantica and intermediate Fasciola in Genbank revealed seven identical variable sites of investigated isolates. Among 19 samples, 12 individuals had ITS-2 sequences completely identical to that of pure F. hepatica, five possessed the sequences identical to F. gigantica type, whereas two shared the sequence of both F. hepatica and F. gigantica. No variations in length and nucleotide composition of ITS-2 sequence were observed within isolates that belonged to F. hepatica or F. gigantica. At the position of 218, five Fasciola containing a single-base substitution (C>T) formed a distinct branch inside the F. gigantica-type group which was similar to those of Asian-origin isolates. The phylogenetic tree of the Fasciola spp. based on complete ITS-2 sequences from this study and other representative isolates in different locations clearly showed that pure F. hepatica, F. gigantica type and intermediate Fasciola were observed. The result also provided additional genetic evidence for the existence of three forms of Fasciola isolated from native cattle in Korea by genetic approach using ITS-2 sequence.