It’s been a frustrating time over the last few weeks in the lab trying to amplify, clone and sequence aquatic invertebrate flaviviruses from holothurian samples. Earlier, we found that we could successfully amplify (by PCR) a product of expected size from at least one Apostichopus (Parastichopus) californicus sample collected in 2016. However, upon cloning (and we’ve had some problems, unexpectedly, even being able to generate plasmids from clones) and sequencing (often failing… still trying to understand what has changed in the lab, since we’ve always had good success) we found that this amplicon, while correctly sized, was not a flavivirus sequence. Frustrated at the slow progress and difficulty in sequencing aiFVs, we took two approaches. First, we designed quantitative reverse transcriptase PCR (qRT-PCR) primers/probes around two regions of the sea cucumber flavivirus genome – the nonstructural region 5 (NS5, which is otherwise known as the RNA-dependent RNA polymerase), and the putative envelope regtion of the genome. We then applied these to 48 samples of A. californicus cDNAs prepared in 2018 for another project, and to our excitement one of these samples came up positive for the envelope primer/probe set (but not the NS5 primer/probe set). So we took this sample (which was collected in Ketchikan, Alaska, in February 2017) and attempted amplification with our original PCR primers targeting NS5/RdRp, and generated an amplicon of expected size! However, based on past failures, we didn’t get our hopes up. So we cloned and sequenced these products, and today received the exciting news that we had the expected sequence – in other words, we now have a PCR-based assay for aiFVs targeting NS5, and a qPCR assay targeting the envelope region- hooray! We have been waiting for this for the last 5 months.
So where to now? We have in our sample archive ~50 sea cucumber tissue samples amenable to RNA work. So we’ll be extracting these, amplifying them with these primer/primer-probe sets, and seeing what lights up. Soon we will also solicit scientists on the west coast for sea cucumber samples as part of our wider phylogenetic survey. And we will also apply our degenerate, pan-aiFV primer sets against any detected sample to figure out whether this virus is really unique or whether it has some level of [micro] diversity. Fingers crossed – though at least we now know that this virus is actually real and is out in sea cucumber populations!