Apis Cerana Sacbrood Virus Phylogenetic Analysis And Construction Of Infectious Clones

The Sacbrood virus(SBV)is widely distributed in European honey bees,Apis mellifera.AcSBV,a distinct SBV strain in Asian honey bees(A.cerana),causes larva death before pupation and often depopulates colonies,leading to collapse.It is the most severe disease in A.cerana beekeeping.AcSBV infects A.cerana in most natural habitats,yet occurrences were not reported in Taiwan before 2015 and were not a concern for local beekeepers.However,in 2016,A.cerana beekeepers in central Taiwan reported SBV-like symptoms.We screened samples of larvae using RT-PCR and surveyed asymptomatic apiaries in north Taiwan.Phylogenetic analyses suggested that AcSBV isolates from central Taiwan were introduced;all isolates had high similarity in sequences to AcSBV genomes identified in mainland China,Vietnam,and Korea and distinct differences to SBV sequence identified in Taiwan China.The overall prevalence in symptomatic colonies was low.No latent infections were detected in asymptomatic colonies.The AcSBV epizootic may not yet have reached its highest potential.Sacbrood virus(SBV)causes destructive disease in honey bees.It belongs to Iflaviridae,a single positive-stranded RNA virus encoding an ORF that translates into a polyprotein cleavage in a host cell.Single ORF with no experimental reverse genetic data has made it difficult to create an infectious clone with desired mutations or a reporter gene.In this study,we constructed a bi-cistronic SBV clone that imitates dicistrovirus genome structure,two ORFs divided by intergenic region IRES(IGR-IRES).Black queen cell virus IGR-IRES was cloned and integrated at the end of 3'nontranslated region(NTR)of the cloned Apis cerana SBV genome,followed by enhanced green florescence protein(EGFP)gene and poly(A)tail.After transcription,viral RNA was injected into larvae to produce the cloned virions,which were used as oral inoculum for two more generations.The oral inoculated larvae simultaneously expressed typical sacbrood symptoms and EGFP fluorescence,which demonstrated that the bi-cistronic SBV clone is stable and functionally similar to wildtype SBV.In addition,the results suggested SBV 3'NTR does not affect the IGR-IRES induced expression and the replication of the virus.Our SBV clone with easy to trace EGFP expression can lead to better understanding through reverse genetics,pathogenesis,and transmission mechanisms of SBV in A.cerana.In addition,SBV shares the same genome structure and high similarity in sequences with other iflaviruses in honey bees and other hosts,and the same method may be easily applied to those viruses.