After a frustrating several months of trial and error, the team is back in action looking at sea cucumber aiFVs. We now have a validated method of detecting aiFVs in sea cucumber tissues – which involves the following steps:
- Tissue collection, preservation, RNA extraction and copy DNA (cDNA) synthesis
- Quantitative reverse transcriptase PCR (qRT-PCR) targeting the putative envelope (ENV) region of the Apostichpous (formerly Parastichopus) californicus associated flavivirus (PcaFV). This assay was validated against a suite of sea cucumbers and sequencing of amplicons.
- Amplification of qRT-PCR positive samples using two primer sets targeting: 1) the PcaFV nonstructural 5 (NS5) region, and 2) primers targeting the same region but with base degeneracies to amplify similar flaviviruses. These have been validated against sea cucumber samples as well as a synthetic gene fragment.
So we are go for our next (or technically first) phase of the sea cucumber project! Over the last 6 years the lab has collected Apostichopus californicus samples from southeast Alaska and the Salish Sea, and will be running these through this pipeline, which ultimately will lead to understanding aiFV prevalence and diversity. An early result – in screening 20 samples from southeast Alaska (from 2017), PcaFV (based on ENV-qRT-PCR) was detected in 4 samples, meaning around a 20% prevalence. And within different organ systems, the virus shows up mostly in respiratory tree tissues. However, this is a tiny number of samples, so we will look to analyze a wider suite in the coming months.
BUT – a big limitation on our work is lack of samples! Currently we do not anticipate being able to sample A. californicus at field sites until the fall (due to COVID). However, if any scientist in CA, OR, WA or AK (or BC!) is able to do so, we would love to collaborate – please get in touch! Jay and Chris will be working on this project starting next week.
Meanwhile, we are also starting to work on a rapid and field-deployable protocol for PcaFV based on isothermal amplification (RT-LAMP) and colorimetric (gold) reporting. The idea is that we will be able to deploy this to local high schools, the fishery and other groups in southeast Alaska to monitor for PcaFV over time. This is essentially a new approach for our lab, so stay tuned for some interesting developments.
Finally, our work on seagrass viromes is going at full speed. So far we’ve sequenced viromes from a variety of Zostera samples from New Zealand, Japan, and Cape Cod (USA). However, we’re still looking for collaborators elsewhere in the world to understand the distribution of the really interesting viruses we have detected in viromes. Not to give everything away, but let’s just say we’re finding evidence of classically fungal- and metazoan-vectored viruses, including those which cause chlorosis in terrestrial plants. If you’re working on seagrasses, or seagrass wasting disease, please get in touch! We’d love to collaborate to see how these viruses map onto other known pathogens (like Labyrinthula zosterae).