Genetic characterization of VP1 gene of seven Sacbrood virus isolated from three provinces in northern China during the years 2008-2012.

The genetic diversity of seven northern China isolated Sacbrood virus strains (SBV) has been analyzed, and hypervariable regions of the VP1 gene of 7 SBV were sequenced and characterized, in order to obtain epidemiological and immunological information, and to suggest typing criteria for SBV. Sequence analysis of hypervariable regions of the VP1 gene in the genome of these isolates revealed a sequence homology of 91.0-99.3% among all seven local SBV isolates from Apis cerana from China, with a similarity of 93.3-100.0% in deduced amino acid sequences. These local isolates shared 87.4-92.8% sequence homology with six SBV reference strains in GenBank (including two SBV reference strains from Apis cerana from China), which represents a 91.8-97.6% similarity in deduced amino acid sequences. Genetic analysis also showed that five SBV strains from Apis cerana from China had a 13-amino-acid deletion at amino acid positions 287-299, and two SBV strains infecting the Korean honeybee had a 17-amino-acid deletion at amino acid positions 284-300 in comparison with other SBV. Phylogenetic analysis revealed two major groups (AC genotype SBV infecting Apis cerana and AM genotype SBV infecting Apis mellifera). The AC genotype could be further divided into subgroups. Based on the results of phylogenetic analysis, a similarity scan of SBV nucleotide sequences was carried out by using Simplot software and results in similar results. Our results suggest possible typing criteria for SBV based on the phylogenetic tree and sequence homology, and also that the virus has host specificity and regional variations.

TaqMan MGB probe fluorescence real-time quantitative PCR for rapid detection of Chinese Sacbrood virus.

Sacbrood virus (SBV) is a picorna-like virus that affects honey bees (Apis mellifera) and results in the death of the larvae. Several procedures are available to detect Chinese SBV (CSBV) in clinical samples, but not to estimate the level of CSBV infection. The aim of this study was develop an assay for rapid detection and quantification of this virus. Primers and probes were designed that were specific for CSBV structural protein genes. A TaqMan minor groove binder (MGB) probe-based, fluorescence real-time quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed; specificity was high and there were no cross-reactivity with healthy larvae or other bee viruses. The assay was applied to detect CSBV in 37 clinical samples and its efficiency was compared with clinical diagnosis, electron microscopy observation, and conventional RT-PCR. The TaqMan MGB-based probe fluorescence real-time quantitative PCR for CSBV was more sensitive than other methods tested. This assay was a reliable, fast, and sensitive method that was used successfully to detect CSBV in clinical samples. The technology can provide a useful tool for rapid detection of CSBV. This study has established a useful protocol for CSBV testing, epidemiological investigation, and development of animal models.