Woah – 2 months flies by! It’s not that we haven’t been working really, really hard in the lab, it’s that we’ve just had so little time to update everyone on the team’s work. After returning from Seattle and adventures in the Puget Sound, Ian and Elliot’s feet were barely on the ground before we started lab work on the samples. First up, Ian extracted DNA from both the sediment samples and plankton tow samples we collected during the cruise, then applied our SSaDV detection protocol. Interestingly, looks like not a lot there, if anything. Hardly a surprise given recent findings* scroll ahead to see what we’re thinking.
Attack of the giant sea star papillae!
Next, Ian turned his attention to the historical samples provided by the California Academy of Sciences and the LA County Museum of Natural History last year. As a bit of background here, we had previously applied the SSaDV detection protocol (using TaqMan qPCR targeting two loci) to 66 samples from 1923 to present day, in which we reported detections as far back as 1942. This led to the interesting and perplexing result that the virus had been around for over 70 years, but only recently has it become a problem. Seeking to further resolve this by cloning out the entire genome and studying it’s sequence heterogeneity, Ian and assistant Chaunte applied this approach to a further 125 samples, representing mainly Pycnopodia, Pisaster ochraceus and Evasterias troscheli from 1905 to present day. The idea was to first detect what samples had detectable SSaDV in them, then use conventional PCR to obtain amplicons of 5 loci across the genome (called NS1, VP4, VP1, and intergenic spacers between NS1 and VP4 and between VP4 and VP1). qPCR identified quite a few samples that were SSaDV positive, including those again from 1942. Applying conventional PCR to these positive samples yielded amplicons, however, in only 3 samples: a P. ochraceus from 1945, a P. ochraceus from 2012 and a P. ochraceus from 2015 (which we put in to make sure our detection protocols actually work). However, only one of the loci amplified from 1945, and only 3 of the 5 from the 2012 sample. When we cloned and sequenced all of these, to our surprise, the 1945 sample, despite having an amplicon of the correct size, was not anything even close to SSaDV, neither were the three loci from 2012! The only reliable detection came from our 2015 sample. Hence, looks like we have to go back on our observation and say that it’s likely something NEW* which shares features with older viruses.
Ian’s also been working with samples sent to the lab (very generously) by Jan Kocian. Earlier this year, we performed a very thorough and fine-scale analysis of tissue specificity of SSaDV, and found that it is present primarily in digestive tissues and external tissues. However, this analysis was performed on animals that were infected and asymptomatic. What we lacked were dead and decomposing animals to see if the patterns observed in the asymptomatic stars held up. And boy did they, and they also provided some very good data on what happens to SSaDV as animals waste. Not only does SSaDV appear to be associated with lesions on the body wall of the animal, but it also appears that the virus causes degradation of surface tissues which in turn facilitates maybe their spread. We’ve now got some new hypotheses that the team is excited to test this spring and summer.
Nasty, wasted sea star. Something’s missing from this specimen…
More animal parts. This is why we wear scrubs in the lab.
Next, Ian performed the exact same analysis as the historical work against 126 samples of sea stars collected from diverse geographical locations, including South Africa, Chile, Taiwan, Hong Kong, the Great Barrier Reef, Yellow Sea, Okinawa, and also samples from the east coast of the US. Several samples (only 9) were positive for SSaDV by qPCR. Attempting the same cloning strategy, it became clear that a virus similar to SSaDV (something like 82 – 85%) was in the samples from China and the Gulf of Maine, as well as more distant viruses from Okinawa and South Africa. The China and Gulf of Maine stars are significant, since there has been wasting there. 82 – 87% nucleotide identity is pretty darned close! Ian is working with student Philip Fargo to unravel viral metagenomes prepared from these same samples.
Where is this? Hint: it’s not on the west coast of the US…
Meanwhile, in the lab, Elliot’s been hard at work extracting DNA from a variety of samples for bacterial and viral analyses. He’s been coordinating a team of undergrads, including Mitch Johnson (who was on the cruise), Chaunte Lewis, and newcomer Victor Biguma, to get all the cruise samples extracted. At the same time, we’ve also worked on samples sent to us from a variety of places, including the New England Aquarium in Boston where they have recently experienced SSWD.
So lots going on! We will update this website in another couple of months as the research progresses…
Hockey goalie: It’s a great way to train for the peer review process.