Meta-transcriptomic Analysis Of Seahorses With Their Live Feed And The Discovery Of Novel RNA Viruses

With the extensive use of meta-transcriptome sequencing technology in virome studies in recent years,more and more RNA viruses have been discovered.Seahorses are one of the important species in the aquaculture industry due to their medicinal and ornamental value.Brine shrimp(Artemia),which is the starter feed for a variety of aquaculture species such as seahorses,is also very important in the aquaculture industry.However,the current virome studies on seahorses and brine shrimp are not sufficient.The aim of this study was to conduct virological studies on seahorses and brine shrimp based on the relationship of the food chain,so as to correlate and expand the species coverage of virome studies in aquaculture and the viral diversity of related species,and to identify viruses that may be a potential threat to the seahorse aquaculture industry.This study characterized the novel RNA viruses found in this study based on six lnc RNA libraries generated from three diseased and three normal lined seahorse(H.erectus)samples collected in 2019,and 87 lnc RNA libraries generated from a total of 141 batches of Artemia cysts samples collected successively from 1977 to 2019 from six continents around the world.In this study,a total of 539 virus-associated contigs were screened in lined seahorse samples by meta-transcriptomic analysis,and three novel viruses from the Astroviridae,Paramyxoviridae,and Picornaviridae families,respectively,were finally identified.Among them,the newly identified astrovirus had genomic structural differences compared with known astroviruses,and the discovery of this virus increased the genomic structural diversity of astroviruses.In addition,we identified for the first time a strain of nervous necrosis virus in the lined seahorse.At the virome level,diseased samples showed higher viral diversity and viral abundance compared to normal samples,and these results reveal the diversity and cross-species transmission of viruses in lined seahorse and highlight the potential threat of viruses to the lined seahorse.In addition,this study performed meta-transcriptomic analysis combined with reverse transcription-polymerase chain reaction(RT-PCR),5’/3’ rapid-amplification of c DNA ends(5’/3’ RACE),and Sanger sequencing,we identified a novel virus with a special genomic structure in Artemia cysts,brine shrimp virga-like virus 1(Bs VV1),which is widespread in the Artemia cysts worldwide,and obtained the complete genome of the virus.While novel viruses identified in previous studies generally have a certain similarity to the genome structure of known related viruses,surprisingly,different genomic regions of this novel virus identified in Artemia cysts were annotated to members of the Virgaviridae and Bunyavirales,respectively,with virgavirus being positive-sense viruses and bunyavirus being negative-sense viruses,which have completely different translational patterns.In addition,phylogenetic analysis of Bs VV1 in Artemia cysts collected from different regions in this study showed a significant correlation of this virus in terms of phylogeny with geographical location.To investigate whether Bs VV1 could proliferate in Artemia cells,we have also developed Artemia primary cells using Artemia cysts and constructed a highly sensitive and specific quantitative reverse transcription PCR(RT-q PCR)assay for the detection of Bs VV1 based on the conserved domain of the virus.Since Artemia are a species hundreds of millions of years old and Bs VV1 have been found in Artemia cysts from multiple locations worldwide,this new virus from Artemia is important for us to study the evolution of viruses.