Bombyx mori nucleopolyhedrovirus (BmNPV) Bm64 is required for BV production and per os infection.
BACKGROUND
Bombyx mori nucleopolyhedrovirus (BmNPV) orf64 (Bm64, a homologue of ac78) is a core baculovirus gene. Recently, Li et al. reported that Ac78 was not essential for budded viruses (BVs) production and occlusion-derived viruses (ODVs) formation (Virus Res 191:70-82, 2014). Conversely, Tao et al. demonstrated that Ac78 was localized to the BV and ODV envelopes and was required for BV production and ODV formation (J Virol 87:8441-50, 2013). In this study, the function of Bm64 was characterized to determine the role of Bm64 in the BmNPV infection cycle.


METHODS
The temporal expression of Bm64 was examined using total RNA extracted from BmNPV-infected BmN cells at different time points by reverse-transcription PCR (RT-PCR) and 5' RACE analysis. To determine the functions of Bm64 in viral replication and the viral phenotype throughout the viral life cycle, a deletion virus (vBm(64KO)) was generated via homologous recombination in Escherichia coli. Viral replication and BV production were determined by real-time PCR. Electron microscopy was used to detect virion morphogenesis. The subcellular localization of Bm64 was determined by microscopy, and per os infectivity was used to determine its role in the baculovirus oral infection cycle.


RESULTS
Viral plaque and titer assay results showed that a few infectious BVs were produced by vBm(64KO), suggesting that deletion of Bm64 affected BV production. Viral DNA replication was detected and polyhedra were observed in vBm(64KO)-transfected cells. Microscopy analysis revealed that Bm64 was predominantly localized to the ring zone of the nuclei during the infection cycle. Electron microscopy showed that Bm64 was not essential for the formation of ODVs or the subsequent occlusion of ODV into polyhedra. The per os infectivity results showed that the polyhedra of vBm(64KO) were unable to infect silkworm larvae.


CONCLUSIONS
In conclusion, our results suggest that Bm64 plays an important role in BV production and per os infection, but is not required for viral DNA replication or ODV maturation.
Sericultural Research Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China.|Lab of Silkworm Biotechnology, College of Animal Sciences, Zhejiang University, Hangzhou, 310058, China.
Lin
L
Chen
Lab of Silkworm Biotechnology, College of Animal Sciences, Zhejiang University, Hangzhou, 310058, China.
Yunwang
Y
Shen
Lab of Silkworm Biotechnology, College of Animal Sciences, Zhejiang University, Hangzhou, 310058, China.
Rui
R
Yang
Lab of Silkworm Biotechnology, College of Animal Sciences, Zhejiang University, Hangzhou, 310058, China.
Xiaofeng
X
Wu
Sericultural Research Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China.
Wenjun
W
Hu
Sericultural Research Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China. guoxin.shen@ttu.edu.
Guoxin
G
Shen
D000818:Animals; D012831:Bombyx / Q000821:virology*; D002460:Cell Line; D017353:Gene Deletion; D020869:Gene Expression Profiling; D046529:Microscopy, Electron, Transmission; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000502:physiology*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000648:ultrastructure; D057074:Virus Release
D014764:Viral Proteins