Current large-scale recombinant adeno-associated virus (rAAV) production systems based on the baculovirus expression vector (BEV) remain complicated and cost-intensive, and they lack versatility and flexibility. Here we present a novel recombinant baculovirus integrated with all packaging elements for the production of rAAV. To optimize BEV construction, ribosome leaky-scanning mechanism was used to express AAV Rep and Cap proteins downstream of the PH and P10 promoters in the pFast.Bac.Dual vector, respectively, and the rAAV genome was inserted between the two promoters. The yields of rAAV2, rAAV8, and rAAV9 derived from the BEV-infected Sf9 cells exceeded 105 vector genomes (VG) per cell. The BEV was shown to be stable and showed no apparent decrease of rAAV yield after at least four serial passages. The rAAVs derived from the new Bac system displayed high-quality and high-transduction activity. Additionally, rAAV2 could be efficiently generated from BEV-infected beet armyworm larvae at a per-larvae yield of 2.75 ± 1.66 × 1010 VG. The rAAV2 derived from larvae showed a structure similar to the rAAV2 derived from HEK293 cells, and it also displayed high-transduction activity. In summary, the novel BEV is ideally suitable for large-scale rAAV production. Further, this study exploits a potential cost-efficient platform for rAAV production in insect larvae.

Genome-wide association studies (GWASs) discovered a number of SNPs and genes associated with Alzheimer's disease (AD). However, how these SNPs and genes influence the liability to AD is not fully understood. We deployed computational approaches to explore the function and action mechanisms of AD -related SNPs and genes identified by GWASs, including the effects of 195 GWAS lead SNPs and 338 proxy SNPs on miRNAs binding and protein phosphorylation, their RegulomeDB and 3DSNP scores, and gene ontology, pathway enrichment and protein-protein interaction network of 126 AD-associated genes. Our computational analysis identified 6 lead SNPs (rs10119, rs1048699, rs148763909, rs610932, rs6857 and rs714948) and 2 proxy SNPs (rs12539172 and rs2847655) that potentially impacted the miRNA binding. Lead SNP rs2296160 and proxy SNPs rs679620 and rs2228145 were identified as PhosSNPs potentially influencing protein phosphorylation. AD-associated genes showed enrichment of “regulation of beta-amyloid formation”, “regulation of neurofibrillary tangle assembly”, “leukocyte mediated immunity” and “protein-lipid complex assembly” signaling pathway. Protein-protein interaction network and functional module analyses identified highly-interconnected “hub” genes (APOE, PICALM, BIN1, ABCA7, CD2AP, CLU, CR1, MS4A4E and MS4A6A) and bottleneck genes (APOE, TOMM40, NME8, PICALM, CD2AP, ZCWPW1, FAM180B, GAB2 and PTK2B) that created three tight subnetworks. Our results provided the targets for further experimental assessment and further insight on AD pathophysiology.

Neurotropic viral transsynaptic tracing is an increasingly powerful technique for dissecting the structure and function of neural circuits. Herpes simplex virus type 1 strain H129 has been widely used as an anterograde tracer. However, HSV tracers still have several shortcomings, including high toxicity, low sensitivity and non-specific retrograde labeling. Here, we aimed to construct high-brightness HSV anterograde tracers by increasing the expression of exogenous genes carried by H129 viruses. Using a Trojan horse-like strategy, a HSV/AAV (adenoassociated virus) chimaera termed H8 was generated to enhance the expression of a fluorescent marker. In vitro and in vivo assays showed that the exogenous gene was efficiently replicated and amplified by the synergism of the HSV vector and introduced AAV replication system. H8 reporting fluorescence was brighter than that of currently available H129 tracers, and H8 could be used for fast and effective anterograde tracing without additional immunostaining. These results indicated that foreign gene expression in HSV tracers could be enhanced by integrating HSV with AAV replication system. This approach may be useful as a general enhanced expression strategy for HSV-based tracing tools or gene delivery vectors.