HearNPV Pseudotyped with PIF1,2,and 3 from MabrNPV：Infectivity and Complex Stability

Effective oral infection is set off by interaction of a group of conserved per os infectivity factors(PIFs) with larval midgut columnar epithelial cells. We constructed pseudotyped viruses by substituting pif1, pif2 or pif3 genes of Helicoverpa armigera nucleopolyhedrovirus(Hear NPV) with their homologs from Mamestra bracissae multiple nucleopolyhedrovirus and tested their infectivity to tissue culture cells and to larvae. Transfection and infection assays revealed that all recombinant viruses generated infectious budded virus in both cell culture and in larvae. Electron microscopy showed synthesized occlusion body and occlusion derived virus(ODV) were morphologically indistinguishable from those of the parental virus. By contrast, feeding assays revealed that pseudotyped viruses could not rescue oral infectivity except for pif3 pseudotyped virus that only partially rescued oral infectivity but at a mortality rate much lower than that of the parental Hear NPV. Consistent with the bioassay result, PIF complex was detected in ODVs of pif3 pseudotyped virus only but not in pif1 or pif2 pseudotyped viruses. Our results suggest that PIF complex is essential for oral infectivity, and in the formation of the PIF complex, PIF1, 2 are virus-specific while PIF3 does not appear to be as specific and can function in heterologous environment, albeit to a much more limited extent.

Genomics and proteomics of Apis mellifera filamentous virus isolated from honeybees in China

Apis mellifera filamentous virus(Am FV)is a large DNA virus that is endemic in honeybee colonies.The genome sequence of the Am FV Swiss isolate(Am FV CH–C05)has been reported,but so far very few molecular studies have been conducted on this virus.In this study,we isolated and purified Am FV(Am FV CN)from Chinese honeybee(Apis mellifera)colonies and elucidated its genomics and proteomics.Electron microscopy showed ovoid purified virions with dimensions of 300–500×210–285 nm,wrapping a 3165×40 nm filamentous nucleocapsid in three figure-eight loops.Unlike Am FV CH–C05,which was reported to have a circular genome,our data suggest that Am FV CN has a linear genome of approximately 493 kb.A total of 197 ORFs were identified,among which36 putative genes including 18 baculoviral homologs were annotated.The overall nucleotide similarity between the CN and CH–C05 isolates was 96.9%.Several ORFs were newly annotated in Am FV CN,including homologs of per os infectivity factor 4(PIF4)and a putative integrase.Phylogenomic analysis placed Am FVs on a separate branch within the newly proposed virus class Naldaviricetes.Proteomic analysis revealed 47 Am FV virionassociated proteins,of which 14 had over 50%sequence coverage,suggesting that they are likely to be main structural proteins.In addition,all six of the annotated PIFs(PIF-0–5)were identified by proteomics,suggesting that they may function as entry factors in Am FV infection.This study provides fundamental information regarding the molecular biology of Am FV.