The development of a laser microcapture method for isolating single infectious nucleopolyhedrovirus occlusion bodies from suspensions

Abstract

Single occlusion body (OB) infection studies provide crucial insight into the virulence and transmission of nucleopolyhedroviruses (NPVs). Current methods of NPV infection are based on end-point dilution and are therefore subject to dose errors. This study aimed to develop a laser capture microdissection (LCM) technique to isolate single infectious NPV OBs from suspensions. Helicoverpa armigera nucleopolyhedrovirus (HearNPV) OB suspensions were spotted onto membrane slides with an erioglaucine solution, which was found to increase the contrast of the OBs. When viewed using a light microscope, the OBs appeared to be round, with an average size of 1.10 μm (standard error of the mean= 0.11 μm) and were characterised by a dark solid outline. Single OBs were laser catapulted into the caps of 0.5 ml PCR tubes. Post-microdissection examination of the caps revealed the presence of an excision containing a single OB. To assess the in vivo infectious properties of single LCM-isolated OBs, the frequency of lethal infection with single OBs was assessed using second instar Helicoverpa armigeralarvae and was found to be 4.83%. To determine whether the LCM method of infection follows a typical dose-mortality response, the slope of the probit-log dose regression line obtained was compared to that of droplet feeding and diet contamination. The slopes were not significantly different for the different bioassay methods, indicating that LCM does not affect the infectious properties of the OBs. To assess the suitability of DNA extracted from LCM-isolated OBs for downstream amplification, PCR amplification was conducted using DNA extracted from 1, 10 or 100 OBs. Resolution of the PCR amplicons of a core baculovirus gene, me53, indicated that the gene was successfully amplified in two of the eight single OB samples, and all of the samples where DNA was extracted from larger numbers of isolated OBs. This indicated that LCM had no inhibitory effects on DNA extraction or amplification and that amplification failure in the remaining single OB samples was likely a result of the concentration of template DNA being below the amplification limit. The LCM method developed in this study provides empirical evidence of the number of OBs isolated and could therefore be used to facilitate highly precise single OB studies