Delivered by Ingenta to: Indian Association for the Cultivation of Science (IACS) IP : 202.54.54.240 Mon, 10 Aug 2009 04:13:43 Copyright © 2009 American Scientific Publishers All rights reserved Printed in the United States of America Journal of Nanoscience and Nanotechnology Vol. 9, 5567–5571, 2009 Nanoparticle–Virus Complex Shows Enhanced Immunological Effect Against Baculovirus Ayesha Rahman 1 , Nupur Biswas 2 , Christian Ulrichs 3 , Carmen Büttner 3 , Ratan Lal Bramhachary 1 , Arunava Goswami 1 ∗ , and Alokmay Datta 2 1 Biological Sciences Division, Indian Statistical Institute, 203, B. T. Road, Kolkata 700108, India 2 Suface Physics Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064, India 3 Humboldt-Universität zu Berlin, Institute for Horticultural Science, Lentzeallee 55/57, 14195 Berlin, Germany Insects protect themselves from majority of infections by a non-specific innate immune system (present in both vertebrates and invertebrates). Bombyx mori nuclear polyhedrosis virus (BmNPV), a baculovirus, causing the deadly grasserie disease is a scourge to silkworm industry and we report here the first success in combating this disease with the help of a nanosilica–virus complex. Hydrophobic aluminium silicate nanoparticles were mixed with live BmNPV in vitro. This mixture was injected into one day old 5th instar silkworm larvae (into the hemocoel at the third abdominal spir- acle) before challenging the larvae with live BmNPV via a second injection. This led to substantial enhancement of longevity in the diseased silkworm larvae and 35 ± 53% larvae completed their life- cycle (i.e., formed normal pupae and enclosed as moth). On the other hand, 100% larvae infected with BmNPV alone died within 36 hours. The larvae treated with nanoparticles before infection had a longer lifespan but all of them eventually succumbed, not a single larva metamorphosed to adult stage. Results suggest two pathways of host protective response—one mediated by nanoparticle- alone and the second, more important, via non-specific innate immunological mechanism. AFM and confocal studies show that nanoparticles alter 3-D molecular structure of the virus envelope. Possi- bly this exhibits novel potent epitope(s) which stimulate(s) anti-viral machinery in infected silkworm larvae. SDS-PAGE results suggest that 39 KDa viral protein is the major target of the nanoparticles. Keywords: BmNPV, Nuclear Polyhedrosis Virus, Nanoparticle, Bombyx mori, Grasserie Disease, Insect Immunity. 1. INTRODUCTION Bombyx mori nuclear polyhedrosis virus (BmNPV) is a circular double stranded DNA virus belonging to the family baculoviridae. The pathogenicity of baculovirus is confined to the invertebrates, especially in lepidopteran insects. In nature, infection occurs when occluded bodies (OBs) are ingested by silkworm larvae via oral route. OBs in the milieu of alkaline gut juice (pH 9.5–11.5) release polyhedra derived virion (PDV). PDVs enter the cells fus- ing directly with the plasma membrane of the microvilli. 1 2 Neutralization of BmNPV infection using anti-PDV anti- bodies has been demonstrated implying that the process of attachment and entry might be receptor mediated. 3 BmNPV causes primary infection in the midgut colum- nar epithelial cells followed by a systemic lethal spread in a number of silkworm organ tissues where progeny OBs ∗ Author to whom correspondence should be addressed. are formed. OBs can withstand adverse environments and reestablish infection cycle when OBs from dead host con- taminate silkworm feed. 3 Penetration of the peritrophic membrane by PDV (after mid gut infection) is known to be enhanced by a proteinaceous viral factor. 4 In cell culture, 24 hours post infection, a large number of bud- ded virions (BVs) leave the host nuclei via budding of the nuclear membrane. BVs are responsible for the spread of infection in different tissues of silkworm. 5 BVs are released in the hemocoel and the generalized infection pro- cess ensues. At the later stages of infection, BV produc- tion terminates. In stead, PDVs are produced, occluded in the OBs and released from dead larvae to the environ- ment. Until now, no specific antiviral antidote, therapeutic or prophylactic, exists. In recent years, depending on the tunability of physico-chemical properties (such as surface chemistry, size, solubility etc.), nanoparticles have been used in numerous biomedical applications from targeted drug/antibody delivery to imaging. 6 7 Here we show that J. Nanosci. Nanotechnol. 2009, Vol. 9, No. 9 1533-4880/2009/9/5567/005 doi:10.1166/jnn.2009.1198 5567