Jian Yang,Jia Wang,Xiaowei Peng,Chengfeng Lei,Xiulian Sun,Jia Hu
Dendrolimus punctatuscauses great damage to pine forests worldwide. Dendrolimus punctatus cypovirus 1 (DpCPV-1) is an important pathogen ofD. punctatus. However, the mechanism of DpCPV-1 cell entry has not been elucidated. In this study, we revealed that both GTase and MTase domains of VP3 (B-spike) and VP4 (A-spike) of DpCPV-1 interacted with the midgut proteins ofBombyx mori. Binding and competition assays revealed that GTase, MTase and VP4 played roles as viral attachment proteins. Far-Western blotting and LC-MS/MS analyses identified that heat shock protein 70 (BmHSP70), glutamate dehydrogenase (BmGDH), and angiotensin-converting enzyme (BmACE) in the midgut proteins as ligand candidates of the viral attachment proteins, and this was further verified by co-immunoprecipitation and fluorescence co-localization assays. Viral binding to the host midgutin vitrowas inhibited by pre-treatingB. morimidgut proteins with anti-BmHSP70, anti-BmGDH, anti-BmACE antibodies singly and in combination. Incubating DpCPV-1 virions with prokaryotically expressed BmHSP70, BmGDH, and BmACE also decreased viral attachment to the host midgut.In vivobioassays revealed that viral infection inHelicoverpa armigerawas partially neutralized by BmHSP70, BmGDH, and BmACE. Taking together, we concluded that HSP70, GDH, and ACE mediate DpCPV attachment and entry via binding to the viral attachment proteins, VP3 and VP4. The findings provide foundation for further understanding the entry mechanisms of cypoviruses.
Chaolin Jiang,Xingchen Huo,Lingjie Tang,Meidi Hu,Chunrong Yang,Daji Luo,Jianguo Su
Grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) results in significant economic losses to the global grass carp aquaculture industry. Oral vaccination is an ideal choice for disease precaution in aquaculture. However, oral vaccine can be degraded in the gut. Therefore, the selection of loading materials is essential. In this study, the S6 and S7 fragments (encoding the outer capsid protein VP4 and fibronectin VP56 of GCRV) and grass carp interferons (IFNs), including IFN1, IFN3, and IFNγ2 were used to create DNA vaccines and adjuvants based on pcDNA3.1, respectively. The oral DNA vaccine was encapsulated in poly(lactic-co-glycolic acid) (PLGA) and polyvinyl alcohol (PVA) with IFNs. The PLGA-PVA (PP) nano-microspheres were prepared by double emulsion-solvent evaporation technique. Using transmission electron microscopy and dynamic light scattering assays, it was determined the vaccines had a spherical structure with uniform particle size (643.5 ± 35.3 nm). The nano-microspheres possessed excellent encapsulation efficiency (81.6 ± 2.6%) and loading rate (0.54 ± 0.02%), and simultaneously exhibited negligible hemolytic activity and cell toxicity. The protection rate and tissue viral loads post-GCRV challenge in grass carp were assessed. The oral PP nano-microsphere with pVP4 + pIFN1 (PP41) vaccine increased protection rate by 44% compared with the control group and was correlated with relatively low viral loads in the spleen, head kidney, and hindgut. Further, three crucial serum biochemical indexes, total superoxide dismutase (TSOD), complement C3 (C3), and lysozyme (LZM), were also dramatically increased. Furthermore, mRNA expressions of representative immune-related genes (IgM, IFN1, IFNγ2, MHC-Ⅰ, and CD8α) in the head kidney and spleen were significantly enhanced. In addition, mRNA expression of IgT was significantly boosted in the hindgut. The results indicate that DNA vaccine capsulated with PP is effective against GCRV infection. The present study provides insights into a prospective strategy for oral vaccine development in aquaculture.
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