Development and Validation of a Passive Surveillance System for Early Detection of Pepino Mosaic Virus in Commercial...

The use of environmental nucleic acid (eNA), both DNA and RNA, as a means for surveillance has been a fixture in the scientific literature for many years. The application of environmental screening for genomic signatures of organisms of interest-particularly those of diagnostic concern-is a promising yet still under-utilised tool for sample screening. While the literature tends to focus on the use of high-throughput sequencing (HTS) to detect organisms of interest using metagenomic or metatranscriptomic sampling, this approach is not cost-competitive with more traditional targeted molecular test methods. Consequently, eNA sampling still has not gained significant traction in practical settings despite its popularity in ecological research. To address these issues in a biosecurity context, we report here the development of a testing protocol to monitor irrigation water for the presence of pepino mosaic virus (PepMV) that also includes an endogenous Sphingomonas control. We employed passive sampling through the immersion of filtering devices into the water system at three commercial growing operations at two time points to collect samples with minimal hands-on effort, while simultaneously developing and validating molecular methods for the recovery of RNA competent for both PCR and high-throughput sequencing. We demonstrate not only the ability to detect PepMV from water collections, but also that the method is robust to the accumulation of non-target material and does not lose signal if viruses are only transiently present in the water system. Finally, we developed a capsid-integrity pre-treatment protocol for differentiating between intact and denatured (non-viable) virus particles during PCR testing. This work presents a low-cost and low-effort technique for proactive screening of commercial greenhouse facilities to facilitate early detection of harmful crop pests and pathogens.