Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Thu, 08 Nov 2018 13:40:21 Midgut-based resistance to oral infection by a nucleopolyhedrovirus in the laboratory-selected strain of the smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae) Kento Iwata, 1 Eric Haas-Stapleton, 1,2 Yasuhisa Kunimi, 1 Maki N. Inoue 1 and Madoka Nakai 1, * Abstract A strain of Adoxophyes honmai resistant to Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV) was established from a field-collected colony by repeated selection. Fifth-instar larvae of this resistant strain (R-strain) had over 66 666-fold greater resistance in terms of 50 % lethal concentration values to oral infection of AdhoNPV than non-selected strain larvae (susceptible for AdhoNPV; S2-strain). In this study, the mechanism of resistance to AdhoNPV was determined in R-strain larvae. An assessment of viral genome replication in AdhoNPV-infected S2- and R-strain larvae by quantitative PCR showed no viral genome replication occurring in R-strain larvae. Transcription of AdhoNPV ie-1, vp39 and polyhedrin genes was also not detected in R-strain midgut cells. Besides, a fluorescent brightener had no effect on AdhoNPV infection in either S2- or R-strain. However, binding and fusion of occlusion-derived virus with R-strain were significantly lower than those of S2- strain. These findings suggest that R-strain Adoxophyes honmai larvae possess a midgut-based resistance to oral infection by AdhoNPV in which midgut epithelial cells are infected less efficiently. INTRODUCTION A nucleopolyhedrovirus (NPV) is a circular double- stranded DNA virus belonging to the family Baculoviridae, which consists of the genera Alphabaculovirus, Betabaculo- virus, Gammabaculovirus and Deltabaculovirus [1]. Baculo- viruses are used as biological control agents to control agricultural pests instead of chemical pesticides because viruses of this family exclusively infect insects and do not cause disease in vertebrates or non-target insect taxa. The process of baculovirus infection starts when host larvae ingest viral occlusion bodies (OBs) [2]. OBs are dissolved in the midgut juice to release occlusion-derived viruses (ODVs). ODVs pass the host chitin–protein matrix [known as the peritrophic membrane (PM)], which lines the midgut and directly binds to midgut epithelial cells. Fusion of the ODV envelope with midgut epithelial cell plasma mem- brane leads to the entry of viral nucleocapsids into the cyto- sol. Nucleocapsids migrate to the nucleus, where viral DNA is uncoated, viral genes are expressed and the viral genome is replicated. Progeny viruses are produced, some of which bud to haemocoel as the budded virus virion phenotype, which spreads infection to other tissues in the host. Resistance of lepidopteran insects to baculoviruses has been reported in some laboratory-selected populations [3–8]. Some field populations of Cydia pomonella in Europe also have acquired resistance against Cydia pomonella granulo- virus (CpGV), which is used as a biological control agent [9–11]. The mode of inheritance of CpGV resistance was different among different resistance populations [12, 13], and the resistance mechanism is still not clear. The smaller tea tortrix, Adoxophyes honmai (Tortricidae, Lepidoptera), is a major pest of tea plants in Japan. This pest has acquired resistance to some chemical pesticides and also to sex pheromone compounds used for mating disrup- tion [14, 15]. In our laboratory, to characterize resistance to a potential baculovirus control agent, a resistant strain (R- strain) of Adoxophyes honmai against Adoxophyes honmai Received 31 October 2016; Accepted 11 December 2016 Author affiliations: 1 Graduate school of Agriculture, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183-8509, Japan; 2 Alameda County Mosquito Abatement District, 23187 Connecticut Street, Hayward, CA 94545, USA. *Correspondence: Madoka Nakai, madoka@cc.tuat.ac.jp Keywords: resistance; baculovirus; nucleopolyhedrovirus; midgut; Adoxophyes honmai. Abbreviations: AcMNPV, Autographa californica multiple nucleopolyhedrovirus; BmNPV, Bombyx mori nucleopolyhedrovirus; CpGV, Cydia pomonella granulovirus; dpi, day post-inoculation; LC50, 50% lethal concentration; NPV, nucleopolyhedrovirus; OB, occlusion body; ODV, occlusion-derived virus; PM, peritrophic membrane; qPCR, quantitative PCR; RT-PCR, reverse transcription PCR; R-strain, resistant strain; R18, octadecyl rhodamine B chlo- ride; RFU, relative fluorescence unit. One supplementary figure and three supplementary tables are available with the online Supplementary Material. RESEARCH ARTICLE Iwata et al., Journal of General Virology 2017;98:296–304 DOI 10.1099/jgv.0.000684 000684 ã 2017 The Authors 296