Summer is just over a month finished in Ithaca – at least from a climatic standpoint. With only 3 weeks to go before classes begin again for students at Cornell, it feels like summer is fading. But you’d never guess it in our lab, where things have been heating up considerably as we pursue both sea cucumber flaviviruses and seagrass microbes!
After a fairly frustrating time trying to study the diversity of aquatic flaviviruses in sea cucumbers – lots of two-steps-forward-one-step-back, and often one-step-forward-two-steps-back results, we made a pretty interesting finding this week. Flaviviruses belong to the family Flaviviridae, and like many single-stranded RNA viruses, their genomes are positive-sense (meaning they run in direction the way that a gene would normally be read by the host gene replication machinery). And like many single-stranded RNA viruses their genome has one large protein-encoding region (which is the code used to make a protein, enzyme, etc). Within this protein-encoding region, there is a region called the RNA-dependent RNA-polymerase (which in flaviviruses is called the non-structural 5 region or NS5. To date, we have been trying to amplify by PCR (which is the same technique used to detect e.g. SARS-CoV-2) one part of the NS5 region, but have met with considerable challenges – either we couldn’t amplify anything, or when we sequenced our PCR amplicons it ended up being something we weren’t intending to amplify (called a spurious amplicon). What was really confusing is that we had developed a new, easy and cheap approach to detect these flaviviruses (called reverse-transcriptase loop-mediated isothermal amplification or RT-LAMP for short) which seemed to validly detect flaviviruses in RNA extracts, at least from what we could tell based on positive controls.
So Jay and Ian went ahead and designed new primers (‘starting positions’) across a large number of regions of the sea cucumber flavivirus, and tried these out against several samples which were RT-LAMP positive, as well as some primers that should detect the RT-LAMP products (for scientists: we used the amplicon as template). Astonishingly, we could detect many other regions of the sea cucumber flavivirus, but NOT the NS5 region. BUT, we could detect the flavivirus NS5 in the RT-LAMP products. A bit confused, we sat down with a genome map of the virus, and it became clear that ‘something’ was wrong with the first 3/4 of the NS5 sequence we were designing primers against; the RT-LAMP targeted the last 1/4 of the region. A ha! Either the flavivirus genome sequence on which our work was based was flawed, or the flavivirus today is different from the flavivirus we recovered in 2016 (i.e. the one we designed our primers off of). So onwards and upwards – next, we will try and amplify the ‘current’ NS5 region based on successful amplicons outside of this region. But the good news is that our RT-LAMP positives, really are positive! And shhhh… we’ve started to work with another species in the southeast Alaska region which has come up with many positives. Stay tuned!
So what percentage of samples are positive that we have tested? We’ve been really fortunate to receive 20 Apostichopus californicus (our primary sea cucumber species) from Ketchikan in June this year, plus 9 of the same species from Friday Harbor Lab (thanks to Kyle Hebert and Megan Schwartz, respectively!!). Of all tissues collected from these, we have around 9% that are positive. This is in addition to several from 2016 (Ketchikan), one from the Salish Sea in 2016, and none from other species collected elsewhere. But this is exactly what we predicted, and bodes well for our future work. If we’d detected the sea cucumber flavivirus in every sample, it would have been problematic since it could have been a persistent virus or perhaps an endogenous (integrated into host genome) element. Still, it also means that for our future experiments we’ll need to test animals in advance to see if they are positive prior to any experiment. In most situations, this is somewhat of an arduous and expensive task – using either qPCR (which requires expensive qPCR instrumentation) or antibody-antigen-based detection (which is expensive and takes a long time to develop. Fortunately, we already have a sensitive, rapid, and easy protocol which is easily deployable: RT-LAMP!
A key focus of our current project is engagement with citizen science, high school students/teachers, fisheries, and indigenous groups to track the flavivirus in the wild. Jay’s been working on a deployable version of the RT-LAMP. What this will involve is sending out a kit to participants with a couple of simple pieces of lab equipment (a water bath, a pipette, pipette tips and a kit of reagents). The actual process will be as follows: collection of a small punch of tissue from sea cucumbers in the wild or after boat retrieval – this doesn’t harm the animals or cause loss of catch value. Then, the participant puts the punch into a tube which they shake for a couple of minutes, after which the participant uses the pipette to transfer a few microliters of fluid into a tube, this gets put into a water bath for 30 mins, and then the participant looks to see if there is a change in color. We’re looking for people to work with – and hope that this will springboard teaching modules in virology (marine viruses and flavivirus replication/pathology) and molecular biology (how DNA amplifies).
Onto our other projects; this week we received a couple of echinoderm samples from the Gulf of Maine, thanks to a commercial collections company. For a couple of days, we had two Asterias forbesi (Forbes’ star; a sea star) and two Cucumaria frondosa (a type of sea cucumber that is common in the North Atlantic). The purpose of receiving these animals was to test our optode, which is used to visualize oxygen. This leads back to our hypothesis that sea stars and sea cucumbers experience hypoxic (no oxygen) conditions at their surface in response to organic matter addition, which may cause tissue damage consistent with e.g. sea star wasting disease. As a first attempt to visualize this, Ian placed specimens of the sea cucumber and sea star immediately next to (within 5mm) of the optode, and looked at imagery over a 4-hour time course. Immediately, it becomes clear that sea stars not only experience hypoxic conditions on their upper (aboral) surface, but also that sea cucumbers experience these conditions less so – probably because they exude less mucus (which supports bacteria and other microorganisms that use up oxygen as they respire), but also because they breathe through a respiratory tree which vents to their anus. Super cool, and it’s gratifying to be able to visualize oxygen around a starfish!
So what’s next for the team? We will be joined this week by Audrey Vinton, a rising senior at Cornell who is working on seagrass wasting disease to perform some molecular work – which we may couple with our new understanding of seagrass viruses to see what best corresponds with wasting lesions in the West Falmouth Harbor. We expect sequences back from new PCR amplicons from the sea cucumber flavivirus sometime early this week. And excited to see what comes from our seagrass aquarium work – Jordan will shortly collect these with Katie Haviland (grad student in Natural Resources) at the West Falmouth Harbor. Dog Days of summer? Perhaps, but it’s raining data cats (and dogs…).