Discovery of Novel Crustacean and Cephalopod Flaviviruses: Insights into the Evolution and Circulation of Flaviviruses between Marine Invertebrate and Vertebrate Hosts

Abstract

Most described flaviviruses (family Flaviviridae) are disease-causing pathogens of vertebrates maintained in zoonotic cycles between mosquitoes or ticks and vertebrate hosts. Poor sampling of flaviviruses outside vector-borne flaviviruses such as Zika virus and dengue virus has presented a narrow understanding of flavivirus diversity and evolution. In this study, we discovered three crustacean flaviviruses (Gammarus chevreuxi flavivirus, Gammarus pulex flavivirus, and Crangon crangon flavivirus) and two cephalopod flaviviruses (Southern Pygmy squid flavivirus and Firefly squid flavivirus). Bayesian and maximum likelihood phylogenetic methods demonstrate that crustacean flaviviruses form a well-supported clade and share a more closely related ancestor with terrestrial vector-borne flaviviruses than with classical insect-specific flaviviruses. In addition, we identify variants of Wenzhou shark flavivirus in multiple gazami crab (Portunus trituberculatus) populations, with active replication supported by evidence of an active RNA interference response. This suggests that Wenzhou shark flavivirus moves horizontally between sharks and gazami crabs in ocean ecosystems. Analyses of the mono-and dinucleotide composition of marine flaviviruses compared to that of flaviviruses with known host status suggest that some marine flaviviruses share a nucleotide bias similar to that of vector-borne flaviviruses. Furthermore, we identify crustacean flavivirus endogenous viral elements that are closely related to elements of terrestrial vector-borne flaviviruses. Taken together, these data provide evidence of flaviviruses circulating between marine vertebrates and invertebrates, expand our understanding of flavivirus host range, and offer potential insights into the evolution and emergence of terrestrial vector-borne flaviviruses.IMPORTANCE Some flaviviruses are known to cause disease in vertebrates and are typically transmitted by blood-feeding arthropods such as ticks and mosquitoes. While an ever-increasing number of insect-specific flaviviruses have been described, we have a narrow understanding of flavivirus incidence and evolution. To expand this understanding, we discovered a number of novel flaviviruses that infect a range of crustaceans and cephalopod hosts. Phylogenetic analyses of these novel marine flaviviruses suggest that crustacean flaviviruses share a close ancestor to all terrestrial vector-borne flaviviruses, and squid flaviviruses are the most divergent of all known flaviviruses to date. Additionally, our results indicate horizontal transmission of a marine flavivirus between crabs and sharks. Taken together, these data suggest that flaviviruses move horizontally between invertebrates and vertebrates in ocean ecosystems. This study demonstrates that flavivirus invertebrate-vertebrate host associations have arisen in flaviviruses at least twice and may potentially provide insights into the emergence or origin of terrestrial vector-borne flaviviruses. FIG 2 Translation of a complete polyprotein depends on highly st...

Emerging viruses in British Columbia salmon discovered via a viral immune response biomarker panel and metatranscriptomic sequencing

“…Interestingly, recent evidence of arthropod viruses circulating in the blood of a vertebrate host (71) suggests that crosskingdom transmission could be more common than previously thought. Similarly, there have been reports of novel flaviviruses infecting both marine vertebrates and invertebrates (72). Here we show, using in-situ hybridisation, that this putative virus is not an environmental contaminant, but appears to be associated 325 with an invertebrate parasite of the salmonid host, raising the possibility that there is an alternative invertebrate host for this virus.…”

“…It is not clear if the localisation reflects the dissemination of the virus from the metazoan eggs in the intestine, or infection of an immature stage of the parasite itself. The detection in fish of viruses that phylogenetically resemble viruses of invertebrates reflects recent work which found the sequence from the same virus in both crabs and sharks (72). It is apparent that in the marine environment, certain groups of viruses are transmitted horizontally between invertebrates and 335 vertebrates, in which case distinction between groups of viruses that infect vertebrates and invertebrates is starting to break down.…”

Exploration of the virome of the European brown shrimp (Crangon crangon)

“…To our knowledge, a small number of viruses belonging to the Flaviviridae, are described in crustaceans, except for the ones described by Shi et al [46] and more recently by Parry and Asgari. The flavi-like viruses described in those studies still need further characterization in order to classify them in the correct family and in the correct genus [47]. The two viruses found during this study were approximately 11 and 22 kb (CcFLV1 and CcFLV2, respectively) long.…”

“…2d). Phylogenetically, CcFLV1 clustered together with the recently discovered Crangon crangon flavivirus and Gammarus chevreuxi flavivirus [47]. These viruses are distantly related to a group of insect-only infecting viruses, (e.g.…”

Unmapped RNA Virus Diversity in Termites and Their Symbionts

“…This virus is highly abundant and has a genome structure typical of members of the arthropod infecting/vectored genus Flavivirus. It is therefore possible that Waxsystermes virus is indeed termite-infecting, although it is impossible to exclude that it is associated with a symbiont or parasite of the termite, and it is striking that Waxsystermes is most closely related to recently discovered marine-vertebrate associated viruses [ 54 ]. As such, establishing the true host of Waxsystermes virus has more broad-ranging implications for our understanding of flavivirus evolution as a whole.…”