Evidence of Apis cerana Sacbrood virus Infection in Apis mellifera

Abstract

Sacbrood virus(SBV) is one of the most destructive viruses in the Asian honeybee Apis cerana but is much less destructive in Apis mellifera In previous studies, SBV isolates infecting A. cerana(AcSBV) and SBV isolates infecting A. mellifera(AmSBV) were identified as different serotypes, suggesting a species barrier in SBV infection. In order to investigate this species isolation, we examined the presence of SBV infection in 318A. mellifera colonies and 64A. cerana colonies, and we identified the genotypes of SBV isolates. We also performed artificial infection experiments under both laboratory and field conditions. The results showed that 38A. mellifera colonies and 37A. cerana colonies were positive for SBV infection. Phylogenetic analysis based on RNA-dependent RNA polymerase (RdRp) gene sequences indicated that A. cerana isolates and most A. mellifera isolates formed two distinct clades but two strains isolated fromA. mellifera were clustered with theA. cerana isolates. In the artificial-infection experiments, AcSBV negative-strand RNA could be detected in both adult bees and larvae ofA. mellifera, although there were no obvious signs of the disease, demonstrating the replication of AcSBV inA. mellifera Our results suggest that AcSBV is able to infectA. melliferacolonies with low prevalence (0.63% in this study) and pathogenicity. This work will help explain the different susceptibilities ofA. cerana and A. melliferato sacbrood disease and is potentially useful for guiding beekeeping practices.

FIG 1

Maximum likelihood consensus tree of SBV isolates. A 386-bp region of the RNA-dependent RNA polymerase gene was used. The tree was constructed with a Tamura 3-parameter distance model. The statistical significance of a particular tree topology is evaluated by bootstrap resampling of the sequences 1,000 times, and bootstrap values of <50% are omitted. Numbers on the nodes indicate clade credibility values. New isolates of A. mellifera (circles) and A. cerana (triangles) described in this study are indicated. Strains are annotated with respect to country of isolation, virus host, and GenBank accession number. Am, A. mellifera; Ac, A. cerana.

FIG 2

Larval mortality rates at 24 h after inoculation (A) and survival rates of adult workers (B) of A. cerana (Ac) and A. mellifera (Am) inoculated with AcSBV under laboratory conditions. In panel A, the boxes represent first quartiles and third quartiles, the horizontal lines indicate the median, and the whiskers represent the minimum and maximum for all observed values. Mortality rates of larvae and survival rates of adult workers were calculated from 20 larvae and 50 adult workers per treatment group, respectively. Am/PBS, A. mellifera larvae fed PBS; Am/AcSBV, A. mellifera larvae infected with AcSBV; Ac/PBS, A. cerana larvae fed PBS; Ac/AcSBV, A. cerana larvae infected with AcSBV. The data are averages from three replicates. *, P < 0.05.

FIG 3

Relative quantification of AcSBV in A. mellifera (A) and A. cerana (B) adult workers inoculated under laboratory conditions. Twelve bees were collected on days 0, 2, 6, and 10 and used for quantification. D, day. **, P < 0.01; ***, P < 0.001.

FIG 4

Detection of negative-strand RNA of AcSBV in A. mellifera larvae and adult workers. Lanes 1, 3, and 5, samples obtained on days 2, 6, and 10, respectively; lanes 2, 4, and 6, negative controls without Tag-SB7f primers; lane 7, negative control without the DNA template. Lanes 1, 3, and 5 in panel A represent pooled samples of 6 bees from each of 3 replicates on each day; in panels B, C, and D, results of one replicate are shown. (A) Adult worker bees challenged with AcSBV in the laboratory. (B) Larvae challenged with AcSBV in the laboratory. (C) Adult worker bees challenged with AcSBV in a colony. (D) Larvae challenged with AcSBV in a colony.