No abstract

In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines.

The gene expression of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was examined in two types of mammalian cells, human HeLa14 and hamster BHK cells. DNA microarray analysis followed by reverse transcription-PCR identified at least 12 viral genes transcribed in both HeLa14 cells and BHK cells inoculated with AcMNPV. 5 rapid amplification of cDNA ends was carried out to examine the transcriptional fidelity of these genes in HeLa14 cells. The transcription of ie-1, ie-0 and gp64 was initiated at a baculovirus early gene motif, CAGT, accompanied by a TATA motif. In addition, the same splicing observed for ie-0 mRNA in Sf9 cells occurred in HeLa14 cells. While the transcription initiation sites for pe38 and p6.9 were not located in the CAGT motif, most of them were in a typical eukaryotic RNA polymerase II promoter structure (a conventional TATA motif and/or an initiator). Interestingly, the expression of ␤-actin was upregulated in the mammalian cells inoculated with AcMNPV. Subsequent experiments using UV-inactivated virus confirmed the upregulation, suggesting that de novo synthesis of viral products is not required for the event. These results indicated that the AcMNPV genome acts as a template for transcription in mammalian cells through the usual infection pathway, though there is no evidence for the functional expression of viral genes at present.Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a type member of the insect-specific virus family Baculoviridae (genus Nucleopolyhedrovirus [NPV]), infects several important pest insects and has been used as a safer biopesticide in Integrated Pest Management program. The nucleopolyhedroviruses are also used as a gene transfer vector in insect cells, providing one of the most efficient systems for the expression of foreign genes in eukaryotic cells (19). Furthermore, recent studies demonstrated that AcMNPV can enter the nucleus of mammalian cells independently of the cell cycle without multiplication (28, 29) and effectively express foreign genes (15) without expressing its own genes (28), suggesting that the baculovirus can be used as a low risk viral vector in the field of gene therapy (10). Recently, AcMNPV was shown to be capable of stimulating production of interferon in mammalian cell lines and when injected into mice conferred protection from lethal infections of encephalomyocarditis virus (13). It was also reported that the intranasal inoculation of a wild-type AcMNPV induced a strong innate immune response, which protected mice from a lethal challenge of influenza virus, through the Toll-like receptor 9 signaling pathway (1, 3). These reports indicate that baculoviruses affects the physiology of mammalian cells though they are not pathogenic to and have no ability to replicate in mammalian cells.NPV is a large rod-shaped virus with a double-stranded closed circular DNA genome (130 to ϳ160 kbp) containing approximately 150 genes (4). NPV genes are usually categorized into four groups: immediate-early, delayed-early, late, and ver...