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Colony Collapse Disorder (CCD), a special case of collapse of honey bee colonies, has resulted in significant losses for beekeepers. CCD-colonies show abundance of pathogens which suggests that they have a weakened immune system. Since honey bee viruses are major players in colony collapse and given the important role of viral RNA interference (RNAi) in combating viral infections we investigated if CCD-colonies elicit an RNAi response. Deep-sequencing analysis of samples from CCD-colonies from US and Israel revealed abundant small interfering RNAs (siRNA) of 21-22 nucleotides perfectly matching the Israeli acute paralysis virus (IAPV), Kashmir virus and Deformed wing virus genomes. Israeli colonies showed high titers of IAPV and a conserved RNAi-pattern of matching the viral genome. That was also observed in sample analysis from colonies experimentally infected with IAPV. Our results suggest that CCD-colonies set out a siRNA response that is specific against predominant viruses associated with colony losses., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A country-wide screen for viral pathogens in Israeli apiaries revealed significant incidence of deformed wing virus (DWV) and Varroa destructor-1 virus (VDV-1). To understand these viruses' possible involvement in deformed wing syndrome of honey bees, we studied their replication in symptomatically and asymptomatically infected bees qualitatively and quantitatively, using RT-PCR, quantitative real-time RT-PCR, and immunodetection of the major viral capsid protein VP1. We found, for the first time, replication of VDV-1 and/or a VDV-1-DWV recombinant virus in the heads of recently emerged symptomatic bees. These viruses replicated to high copy numbers, yielding the major viral capsid VP1 processed for subsequent assembly of viral particles. Our results clearly distinguished between symptomatic and asymptomatic bees infected with VDV-1 and VDV-1-DWV and suggest the hypothesis that VDV-1, in addition to DWV, may be involved in inducing the deformed wing pathology. Thus VDV-1-DWV recombination may yield virulent strains able to cause overt infections in Varroa-infested bee colonies., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Infecting the insect cell line IPLB-Ld652Y with the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) results in global translation arrest, which correlates with the presence of the AcMNPV apoptotic suppressor, p35. In this study, we investigated the role of apoptotic suppression on AcMNPV-induced translation arrest. Infecting cells with AcMNPV bearing nonfunctional mutant p35 did not result in global translation arrest. In contrast, global translation arrest was observed in cells infected with AcMNPV in which p35 was replaced with Opiap, Cpiap, or p49, baculovirus apoptotic suppressors that block apoptosis by different mechanisms than p35. These results indicated that suppressing apoptosis triggered translation arrest in AcMNPV-infected Ld652Y cells. Experiments using the DNA synthesis inhibitor aphidicolin and temperature shift experiments, using the AcMNPV replication mutants ts8 and ts8deltap35, indicated that translation arrest initiated during the early phase of infection, but events during the late phase were required for global translation arrest. Peptide caspase inhibitors could not substitute for baculovirus apoptotic suppressors to induce translation arrest in Ld652Y cells infected with a p35-null virus. However, if the p35-null-AcMNPV also carried hrf-1, a novel baculovirus host range gene, progeny virus was produced and treatment with peptide caspase inhibitors enhanced translation of a late viral gene transcript. Together, these results indicate that translation arrest in AcMNPV-infected Ld652Y cells is due to the anti-apoptotic function of p35, but suggests that rather than simply preventing caspase activation, its activity enhances signaling to a separate translation arrest pathway, possibly by stimulating the late stages of the baculovirus infection cycle.

Spodoptera littoralis cells infected with the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) yielded significantly lower budded virus titers than Spodoptera frugiperda-infected cells and produced very low levels of polyhedrin. Relative to AcMNPV-infected S. frugiperda SF9 cells viral DNA replication was severely reduced in Spodoptera littoralis SL2 cells. Microscopic examination of SL2-infected cells revealed progressive cell blebbing starting at 6-8 hr postinfection and culminating in total cell destruction at 24 hr postinfection. The data suggested that AcMNPV-infected SL2 cells undergo apoptosis. The occurrence of an active apoptotic process in the infected cells was confirmed by: (1) observation of fragmentation of the cell nuclei stained with the specific fluorescent dye DAPI (4',6'-diamidino-2-phenylindole) and (2) the presence of low-molecular-weight DNA oligomers. Neither SL2 cells infected with S. littoralis nuclear polyhedrosis virus (SINPV) nor SF9 cells infected with AcMNPV, respectively, showed nuclear fragmentation or oligonucleosomal ladder formation.

The adeno-associated virus (AAV) rep gene proteins, Rep78 and Rep68, are required for replication of AAV DNA and bind to the AAV replication origin. An AAV genome having a Lys340 to His (K340H) mutation in the consensus purine nucleotide binding site of the rep gene protein exhibited a dominant-negative phenotype for DNA replication. We synthesized both wild-type and the K340H mutant Rep78 protein in a baculovirus expression system. Nuclear extracts of Sf9 cells containing these proteins were examined in gel mobility-shift assays with radiolabeled AAV terminal repeat DNA. Each protein bound specifically to the hairpin configuration of the AAV terminal repeat DNA to yield three shifted components. However the mobility of these components observed with the mutant Rep protein was slightly decreased compared to that with the wild-type Rep78. The addition of an antibody made against an oligopeptide from the carboxyl terminal region of the Rep78 protein generated novel shifted bands in the presence of either extract. Similar results were observed when the wild-type and mutant Rep proteins were expressed from an inducible expression system employing the human immunodeficiency virus type 1 transcription promoter in human 293 cells. These results suggest that the dominant-negative phenotype of the K340H mutation may be mediated by binding of the mutant protein to the AAV replication origin.

When recombinant plasmids containing the entire adeno-associated virus (AAV) genome are transfected into permissive cells infected with a helper adenovirus, infectious AAV particles are efficiently generated. These plasmids can be used to generate mutant AAV genomes or recombinant AAV vectors. Packaging of mutant AAV genomes has required complementation with a second AAV plasmid in the transfection assay which may lead to generation of significant amounts of wild-type AAV recombinants. One approach to alleviate this problem was to generate conditional lethal mutants. We constructed an AAV plasmid recombinant having a nonsense mutation in the AAV rep gene by using oligonucleotide-directed mutagenesis to convert a serine codon to an amber codon. We show that this mutant AAV can be grown on monkey cell lines containing an inducible human serine tRNA amber suppressor. The amber suppression is quite efficient and yields a burst of mutant AAV particles at about 10% of the titer of wild-type AAV. The reversion frequency of the amber mutation appears to be less than 10(-5).

The adeno-associated virus (AAV) rep gene is transcribed from two promoters, p5 and p19, which code for two over-lapping families of rep proteins. The proteins coded by p5 transcripts contain an amino-terminal domain not present in the proteins coded by p19 transcripts. The rep gene is required for AAV DNA replication and also mediates pleiotropic effects in positive and negative regulation of expression of genes driven by either AAV or heterologous promoters. All three functions require rep proteins coded by p5 transcripts. The functions of the rep proteins coded by the p19 transcripts could not be independently discerned since the coding region for these proteins and the p19 promoter are embedded within the coding region of the p5 transcript unit. We describe here an AAV mutant in which the putative AUG initiation codon in the p19 transcripts was altered and which did not express p19-coded rep proteins. This mutant exhibited normal AAV duplex RF DNA replication but was deficient in accumulation of AAV single-stranded progeny DNA and infectious AAV particles. This mutant defines a novel phenotype for a rep gene mutation and suggests a role for the rep proteins in the generation or accumulation of the viral SS DNA. Moreover this mutant distinguishes two different functions of the rep protein(s) in accumulation of the RF molecules and accumulation or processing of the SS DNA molecules, respectively.