The hunt for aquatic invertebrate flaviviruses begins!

The team has started work on our new NSF-funded project looking at aquatic insect-only (aka invertebrate) flaviviruses (aiFVs)! This exciting new direction seeks to understand how environmental variation drives viral replication within sea cucumber tissues. As part of this new endeavor we are trying to find out how widespread and how diverse these aiFVs are by targeted PCR-based analyses – using information gained from prior metagenomic work.

As a bit of background, aiFVs were first observed in terrestrial ecosystems, where they are known to infect insects, distinguishing them from flaviviruses that infect vertebrates via insect vectors. That’s why they’re known as insect-only flaviviruses. In 2016, Shi et al recovered a fairly distant flavivirus resembling these insect-only flaviviruses from the transcriptome of a shark. Subsequent discoveries of related flaviviruses in squid and amphipods broadened the host range of this newly-observed group. Last year we also found a flavivirus genome in the sea cucumber Apostichopus californicus from Southeast Alaska (collected in 2016). And more recently we have found similar genomes in other molluscs and even in Finger Lakes plankton. We’re calling this cluster of similar flaviviruses aiFVs to distinguish them from human pathogens, which include Zika and Dengue viruses.

Phylogenetic represenation of the NS5 region of aiFVs… PcaFV is the sea cucumber flavivirus. It sites within a cluster of sequences that come from aquatic invertebrates and are distinct from those in worms and molluscs… The Tamana Bat virus and Lumpfish virus are both vertebrates which are insectivorous (bat) or graze on parasitic copepods (lumpfish).

Before surveying the diversity of these viruses, we need to design PCR primers which allow amplification in a conserved region (in flaviviruses this is the RNA-dependent RNA-polymerase region (known as NS5). This is no trivial task; the viruses are fairly divergent from one another, and so there are very few regions that share nucleotide identities that enable amplification across all known aiFVs. After searching around, we identified two regions which had sufficient similarity that enabled design of degenerate PCR primers! So efforts are underway to test these new primer sets, starting with samples from 2016 from which we originally obtained the sea cucumber aiFV.

Example of a shared region within the Flavivirus NS5 gene…

Initially, we applied these primers to reverse-transcribed cDNA prepared in 2017, but these did not yield any products on first attempt. There are many reasons this may have happened, including: 1) degraded cDNA template material – this happens with freeze/thaw of extracts; 2) suboptimal PCR conditions – we tried at only one annealing temperature so far, so this needs to be optimized; or 3) incorrect orientation of the primers relative to the packaged strand of this positive sense single stranded RNA virus; the latter isn’t as much of a concern since flaviviruses form a double stranded intermediate during replication, so there would be at least some of both directions in infected cells. We’ll keep trying!

It will work it will work it will work it will work…

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