Molecular, functional and biological characterization of a single enveloped nucleopolyhedrovirus infecting the crop pest Helicoverpa armigera

Abstract

My study involves a detailed molecular and cellular characterisation of a South African isolate of a nucleopolyhedrovints (I.IPV) isolated from the Lepidopteran insect Helicoverpa armigera (HaSNPV-SA). Chapter two entails the in vitro infection of Heliothis zea cell lines with HaSNPV-SA. Within this chapter I optimise the propagation of HaSNPV-SA within the cell-line HzAMI, I obtain a pure enotype (HaSNPV-PI3) and compare the cell-lines HzAMI and Hz2E5 to determine which allows for faster propagation. Within chapter three, I discuss the identification of the ecdysteroid UDP- lucosyltranferase (egt) gene of HaSNPV-SA using a Hz-SNPV gene-specific probe. Studies have shown that the enzyme disrupts the ecdysteroid balance of the host larv4 causing a delay in the onset of melting. That a secreted and active EGT is encoded by HaSNPV-SA was confirmed by assay of infected cell culture medium. Chapter four discusses the diversity of baculovinrs repeated open reading frames (bro) in three nucleopolyhedroviruses of Helicovefpa spp.BROs of some NPV's have been shown to be involved in nucleosome organization that could block cellular replication and/or transcription and switch host machinery to viral DNA or RNA synthesis. I show that the three bro-genes present in four Helicoverpa isolates are not conserved. Furthermore, Northern analyses and/or RT-PCR indicated that all Ha-G4 3 bro-genes were transcribed at either 4hto24h post infection (p.i). The final chapter, chapter five, identifies three essential baculovirus genes helicase, chitinase and late expression (l"fl 8l of HaSNPV- SA and uses them to confirm the placement of HaSNPV-SA in baculovirus phylogeny. All three trees confirm the placement of HaSNPV's as a group II type NPV. Based on sequence similarities, gene placement and conservation and phylogeny it is tempting to say the HaSNPV-SA isolate is a variant of HzSNPV-ELCAR.