Disruption of Bombyx mori nucleopolyhedrovirus ORF71 (Bm71) results in inefficient budded virus production and decreased virulence in host larvae

Abstract

The Bombyx mori nucleopolyhedrovirus (BmNPV) is a baculovirus that selectively infects domestic silkworm. BmNPV ORF71 (Bm71) is not a core set gene in baculovirus and shares 92 % amino acid sequence identity with Autographa californica multinucleocapsid NPV ORF88 (Ac88/cg30). Previously, it has been reported that virus lacking Ac88 had no striking phenotypes in cell lines or host larvae. However, the exact role of Bm71 during BmNPV life cycle remains unknown. In the present study, we constructed a Bm71-disrupted (Bm71-D) virus and assessed the effect of the Bm71 disruption on viral replication and viral phenotype throughout the viral life cycle. Results showed that the Bm71-D bacmid could successfully transfect Bm5 cell lines and produce infectious budded virus (BV). But the BV titer was 10- to 100-fold lower than that of the wild-type (WT) virus during infection, and the decreased BV titer was rescued by Bm71 gene repair virus (Bm71-R). A larval bioassay showed that Bm71-D virus took 7.5 h longer than the WT to kill Bombyx mori larvae. Transmission electron microscopy analysis indicated that the Bm71-D virus-infected cells had typical virogenic stroma, bundles of nucleocapsids and polyhedra. Taken together, these results suggest that Bm71 has important implications for determining BV yield and virulence in viral life cycle even though it is not an essential gene for replication of BmNPV.

Thymosin FromBombyx moriIs Down-Regulated in Expression by BmNPV Exhibiting Antiviral Activity

“…We have constructed the recombinant plasmid pET-28a- BmTHY and preserved it in our lab ( Zhang et al 2012 ). Escherichia coli strains TG1 and BL21(DE3) were cultured in LB medium (5 g of yeast extract, 10 g of tryptone and 10 g of NaCl per liter, pH 7.5) at 37 °C.…”

“…Thymosins are divided into three main groups, α-, β-, and γ-thymosin according to their isoelectric points ( Low and Goldstein 1979 ; Low et al 1979 ). They have been highly conserved during evolution and found in a broad phylogenetic distribution from invertebrates to vertebrates ( Oates et al 1988 ; Oates and Erdos 1989 ; Bodey et al 2000 ; Manuel et al 2000 ; Zhang et al 2012 ). Emerging evidence reveals that vertebrates thymosins have been widely used as pharmacological agents in clinical practice to treat some diseases including immunodeficiency related diseases, chronic hepatitis and certain types of cancers ( Rustgi 2004 ; Cheng et al 2006 ; Kellici and Burazeri 2009 ; Maio et al 2010 ; Kim et al 2012 ).…”

“…Only 18 related proteins composed of thymosin conservative structural domains from lepidoptera can be found in NCBI website ( Holt et al 2002 ; Clark et al 2007 ). These proteins are highly homologous to β-thymosin (Tβ) and are distributed in Drosophila melanogaster, Aedes aegypti, Bombyx mori and other species ( Zhang et al 2012 ). Ciboulot, a homologous thymosin protein characterized in Drosophila , has been implicated in axonal growth and brain metamorphosis ( Boquet et al 2000 ).…”

“…In a previous study, the gene encoding a Bombyx mori thymosin (BmTHY) protein was identified from the cDNA library of silkworm pupae, which is highly homologous to Tβ ( Zhang et al 2012 ). However, the physiological function of BmTHY in Bombyx mori immunity remains unknown.…”

Bombyx mori nucleopolyhedrovirus (BmNPV) Bm64 is required for BV production and per os infection

“…The Bm64 knockout, the repair and the positive control BmNPV bacmids containing polyhedrin and gfp (enhanced green fluorescence protein gene) were constructed by site-specific transposition as previously described [ 34 ]. The pFast-PH-GFP (containing polyhedrin and gfp ) was constructed as described and transformed into electrocompetent DH10BmBac or DH10Bm 64KO cells to generate the Bm64 knockout bacmid (vBm 64KO ) or the positive control bacmid (vBm), respectively.…”