Comparative Study On The Antiviral OsHv-1 Effect Of Shellfish Host Priming By Pathogen-Associated Molecular Patterns (Poly I:C And LPS)

Oyster herpesvirus（Os HV-1）is one of the main prevalent viral pathogens of shellfish,and the virus is lethal to bivalves at rates of 40-100%.At present,Os HV-1 mainly affects shellfish of the ark family,and mass mortality of the ark family is associated with infection by this virus,causing huge economic losses to producers.Due to the mobile water culture model,conventional physicochemical and other control methods are not feasible for the control of viral diseases in shellfish populations.Classical immunology has demonstrated that invertebrates lack B-cell and T-cell mediated adaptive immune processes.However,recent studies have shown that invertebrate innate immune processes are also characterised by a certain"memory".After the priming of invertebrate by pathogen associated molecular pattern（PAMP）,the host,in case of secondary infection,will have a certain anti-viral effect.This phenomenon has opened up a new field for the research of invertebrate immunization.Therefore,it is expected to develop immune prevention and control into a new way of viral prevention and control.Existing studies have shown that Os HV-1infections and the resulting secondary bacterial infections eventually caused the death of shellfish individuals.In this experimental study,ark clams were priming with the PAMPs Poly I:C and LPS,respectively.Depending on the PAMPs and the duration of priming,the priming components were divided into 1DPP,2DPP,7DPP（1,2 and 7 days of Poly I:C priming,respectively）,1DPL,2DPL,7DPL（1,2 and 7 days of LPS priming,respectively）,and the control group was divided into a non-priming group-V（challenge only）and negative control-Control（healthy ark clams without attack and priming）.After priming,the virus was challenged,and the protective effect and duration of these two kinds of PAMPs on antivirus were studied by detecting the change of virus copy number in blood cells of ark clams and counting the survival rate of infected ark clams.By detecting the expression changes of immune-related genes in ark clams after priming and challenge,the mechanism of long-term protection genes of host after priming of Poly I:C and LPS was preliminarily clarified.The related research results enrich the theory of the"memory"of the innate immunity of molluscs.The main findings of the study are as follows.1.Study of the protective effect of Poly I:C and LPS priming on ark clamThe virus copy number in the non-priming group was 10²times higher than that of7DPP,4.3 times higher than that of 1DPP,and 3.4 times higher than that of 7DPL at 3th Days of laboratory infection with Os HV-1.The survival rate of the non-priming group at day 6 was 43%,significantly lower than that of 2DPL-66%,7DPL-70%,1DPP-53%,2DPP-70%and 7DPP-90%.After the attack,the non-priming group started to die on the5th day with a mortality rate of 10%and reached 100%on day 8th,while the 7DPP started to die on day 6 with a mortality rate of 10%and reached 100%by day 10.All of the above findings suggest that Poly I:C and LPS domestication has a protective effect on ark clams.2.Differential changes in immune gene expression between Poly I:C and LPS priming in ark clamsThe genes that may be involved in the long-lasting protective effects of Poly I:C domestication of shellfish have been analysed by transcriptome sequencing.35homologous genes were identified by sequence alignment in the genome and transcriptome of ark clam and two patterns of expression of these genes were identified,namely the sustain expressed genes and the priming-sensitive genes.A total of 35 differential expression genes were screened,among which one was sustained-expression priming type gene and it was a transcription inhibitor with a zinc finger structure.There were 12 priming sensitization genes,of which five were interferon-induced factors;one belonged to an anticancer factor;one low affinity immunoglobulin;one Interferon perlucin;one helicase with zinc finger domain;E3 ubiquitin-ligase;one Double-stranded RNA-specific adenosine deaminase;one Monocarboxylate transporter and one C-type lectin.3.Priming sensitization genes-SbCTL functional testA priming sensitization long-lasting protective gene was first cloned and identified.It is a C-type lectin and has been named SbCTL.The amino acid sequence of SbCTL consists of a signal peptide and a CRD（including 102 amino acid residues）.The amino acid sequence of SbCTL shared 28%-39%similarity with other CTLs.There were two potential Ca²⁺binding sites in SbCTL.The expression of SbCTL m RNA was detected in all selected tissues,with the highest expression in the gills.The expression of SbCTL m RNA increased significantly（P<0.05）with the increase in total Vibrio content at 72 h post-infection.r SbCTL showed the highest binding activity to LPS in ELISA experiments.r SbCTL treatment of ark clam increased the survival rate of ark clam infected with Os HV-1.In summary,SbCTL acts mainly in the late stages of viral infection,i.e.secondary bacterial infection triggered by viral infection,in the immunoprotective effect of SbCTL against Os HV-1 in ark clam.