Analysis Oftranscriptome And Putative Genes Involved In Immunity Of Scapharca Broughtoniiin Responses To Ostreid Herpesvirus-1(OsHV-1)Infection

Blood ark shell(Scapharca [Anadara]broughtonii),also known as hair clams, red shell, blood shell, is a member of Mollusca, Bivalvia, Pteriomorphia, Arcoida, Arcidae, Scapharca. S. broughtoniiis one of the most important marine economic shellfish in Asia, mainly distributed in the Western Pacific, Japan, South Korea, China and the southeast coast of Russia.S. broughtoniiis a kind of large bivalve molluscs preferred living in cold waters, which mainlydistributedin the depths of 3~50 meters. Since 2012,S. broughtoniihas suffered from disease outbreaks mainly caused byOstreid Herpesvirus-1(OsHV-1), which led to the death of a large number percent ofS. broughtonii.This disease has posed a serious threat to the rapiddevelopment of aquaculture. To better understand the pathogenesis of the OsHV-1 to the blood clam, experimental infection was conducted by injecting filtered tissue homogenate into the gastropod of each calm. Experimental infected clams werethen allocated randomly into 3 groups: blank control group, negative contral group andOsHV-1 infected group. After the experiment, the temporal distribution patterns of OsHV-1 loadsin six different tissues were assayed with quantitative PCR(qPCR). No virus was detected in the clams injected with sea water and the negative control tissuehomogenate. While OsHV-1DNA were detected in animals injected with tissue homogenate prepared from naturally infected clams. The virus DNA loads in tissues fluctuated firstly, and then reached about 106 copies viral DNA per ng of total DNA. Pathologicalchanges including lysed connective tissue, dilation of the digestive tubules, eosinophilic inclusion bodies, nuclear chromatin margination and pyknosis were found in affected animals. Transmission electron microscopy(TEM) revealed herpes-like viral particles within the connective tissue of the mantle. These viral particles were about 90-110 nm in diameter with an electron-dense nucleoid packaged in capsid, which resembled those found in naturally infected clams. Overall, this work demonstrated the pathogenesis of OsHV-1 to blood clamsand closely associated with the mass mortality of them. Additionally, our work also indicatedthe stress reaction after injecting OsHV-1 might play a role in the suppression of OsHV-1 replication, while the mechanisms need to be further studied.Related researcheshavebeen carried out for the OsHV-1pathogenicity and epidemiology, but the mechanism of interaction between the virus and S. broughtonii is still very limited. In this study, we characterized the transcriptome of S. broughtonii blood using Illumina paired-end and PacBio RSIIsequencing platform to identify putative genes involved in immunity.A total of 11014 full length cDNA reads and 22312 non full length cDNA reads have been got by using a Pacbio technique. The results were used as a reference sequence, a cDNA library from the blood of health S. broughtonii and OsHV-1 infectedS. broughtoniiwereconstructed and randomly sequenced using theIllumina technique.RNA-seq analysis generated more than 44 million clean paired end(PE) reads, whichwere assembled into 276,997 unigenes(mean size = 200 bp). Based on sequence similaritysearches, 74,529(26.9%) unigeneswere assigned to Gene Ontology(GO) categories, 85009(30.68%)unigeneswere assigned to NCBI non-redundant protein sequences(Nr),32,471(11.72%)unigeneswere assigned to NCBI nucleotide sequences(Nt), 19,611(7.07%) unigeneswere assigned toKEGG ORTHOLOG(KO), 63,132(22.79%) unigeneswere assigned to A manually annotated and reviewed protein sequence database(SwissProt), 73,322(26.47%) unigeneswere assigned toProtein family(Pfam) and 47,653(17.2%)unigeneswere assigned to euKaryotic Ortholog Groups(KOG)respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes pathwaydatabase(KEGG) showed that 19,611 unigenes were mapped to 33 KEGG pathways. Furthermore, we investigated the transcriptome differences of bloodsamples after infection using a tag-based digital gene expression system. We have got 2,998 differential genes, 933 upregulated genes and 2,065 downregulated genes, screening of immune related genes in 1585, mainly including heat shock protein genes, signal transduction genes, apoptosis gene and cytokine.In this study, the sequences and structures of khHSP70, khHSP90, khHbI, khHbIIA, khHbIIB, khAP-1 and immune related genes were analyzed by PacBio.