Submitted to: Baculovirus and Insect Cell Culture Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 1/12/2005
Publication Date: 2/21/2005
Citation: Mcintosh, A.H., Grasela, J.J. 2005. Acmnpv fluorescent gene constructs for stabilization against inactivation by uv light [abstract]. Baculovirus and Insect Cell Culture Conference Proceedings. p. 2.
Technical Abstract: Baculoviruses are rod shaped enveloped insect viruses with a circular double-stranded DNA genome infecting various hosts within the phylum Arthropoda. The family Baculoviridae is comprised of two Genera, the Nucleopolyhedroviruses (NPVs) and the Granuloviruses (GVs). A unique characteristic of members of the Baculoviridae is the production of two virion phenotypes, commonly referred to as budded virus (BV) or extracellular virus (ECV) and occlusion derived virions (ODV). These two virion types mature at different times and sites in the cell during the infection cycle and serve distinctly different functional roles. BV is produced in the late phase of the infection cycle and acquires its envelope by budding through a modified cell membrane and is the primary means of spreading the virus within the hosts and in cell culture. On the other hand, ODV, which is also enveloped, becomes occluded within occlusion bodies (OB) in the nucleus. The OB is comprised of a protein matrix termed polyhedrin for NPVs and Granulin for GVs. OBs are the primary means of spreading the virus from one host to the other in the environment and is thus the type used in applying the baculovirus as a biopesticide to crops and plants. Baculoviruses have been successfully used in the control of numerous insect pests of agricultural crops and forests and include such pests as the corn earworm (Helicoverpa zea), the velvetbean caterpillar (Anticarsia gemmatalis), the diamondback moth (Plutella xylostella), the gypsy moth (Lymantria dispar), the Douglas fir tussock moth (Orgyia pseudotsugata) and the spruce budworm (Choristoneura fumiferana). More recently baculoviruses have been successfully used not only for production of recombinant proteins in insect cells but as vectors for gene delivery in mammalian cells. One of the major disadvantages in employing baculoviruses as biopesticides is their sensitivity to ultraviolet (UV) in the environment with most of the activity being lost within 24 h. Most of the studies addressing this deficiency have employed physical means of protection such as microencapsulation and various additives as protectants such as fluorescent brighteners. In this research, we employed a genetic approach employing genetic manipulation of the AcMNPV genome whereby gene fluorescent proteins could be expressed in the OB protein matrix, viral envelopes of the ODV as well as in the envelope (calyx) surrounding the OB. The genes employed in these studies were the red fluorescent protein (RFP) and the green fluorescent protein (GFP). Occlusion body recombinants carrying these genes were exposed to UV-B light in the laboratory in the chamber of a Suntest CPS system for 2 h. It was determined that 1 min exposure in the Suntest chamber was equivalent to 20 min of exposure to outdoor sunlight. The best protection was afforded by ODV expressing the GFP in its envelope but RFP fused to the polyhedrin protein matrix did provide greater protection against UV-B as compared to wild-type AcMNPV.