Inactivation of Bombyx mori macula-like virus under physical conditions

Abstract

The Bombyx mori macula-like virus (BmMLV) is a member of the genus Maculavirus, family Tymoviridae, and contains a positive-sense single-stranded RNA genome. Previously, we reported that almost all B. mori-derived cell lines have already been contaminated with BmMLV via an unknown infection route. Since B. mori-derived cell lines are used for the baculovirus expression vector system, the invasion of BmMLV will cause a serious safety risk in the production of recombinant proteins. In this study, to determine the inactivation effectiveness of BmMLV, viruses were treated with various temperatures as well as gamma and ultraviolet (UV) light radiation. After these treatments, the virus solutions were inoculated into BmMLV-free BmVF cells. At 7 days postinoculation, the amount of virus in cells was evaluated by real-time reverse transcription PCR. Regarding heat treatment, conditions under 56°C for 3 h were tolerated, whereas infectivity disappeared after treatment at 75°C for 1 h. Regarding gamma radiation treatment, viruses were relatively stable at 1 kGy; however, their infectivity was entirely eliminated at a dose of 10 kGy. With 254 nm UV-C treatment, viruses were still active at less than 120 mJ/cm(2); however, their infectivity was completely lost at greater than 140 mJ/cm(2) UV-C radiation. These results provide quantitative evidence of the potential for BmMLV inactivation under a variety of physical conditions.

Adventitious viruses in insect cell lines used for recombinant protein expression

“…BmMLV has a single linear 6.5 kb genomic RNA encoding 3 putative protein ORFs, and is classified in the Family tymoviridae, genus maculavirus. BmMLV can be inactivated by heat treatment (1h at 75°C), irradiation (10 kGy), and UV light (140 mJ/cm 2 at 254 nm) [52].…”

“…So far, the only cell line that has been clearly shown to be contaminated with BmMLV is the Bm-N cell line, in which the virus was originally discovered [51, 52, 54]. Several silkworm cell lines other than Bm-N have been screened for BmMLV by RT-PCR with primers specific for the capsid protein ORF [51] and western blotting with a polyclonal antiserum against the BmMLV capsid protein [54].…”

Infectious Virions of Bombyx Mori Latent Virus Are Incorporated into Bombyx Mori Nucleopolyhedrovirus Occlusion Bodies

“…Virus titers were expressed as plaque-forming units (PFUs) via plaque assays as described previously [14]. BmLV inoculation was performed as described previously [17]. Student’s t -test was performed with KaleidaGraph computer software version 4.1 (Synergy Software, Reading, PA, USA)…”