Antiviral Functional Study Of Pattern Recognition Receptors Of Kuruma Shrimp Marsupenaeus Japonicus

Shrimp was one of the most important aquaculture species in China that can creat great economic value each year. However, the shrimp aquaculture is always influenced by the disease caused by pathogen such as WSSV and cost a lot of damage. Shrimp mainly rely on the innate immunity to prevent pathogen invading, so the study on shrim immunity, especially the pathogen recognition mechanism is needed.C-type lectins (CTLs) are PRRs that play important roles in immune response. In this study, two new CTLs, Ld1rLecl and LdlrLec2, containing a low-density lipoprotein receptor class A domain (LDLR) were identified and the function and antivial mechanism of LdlrLecl and Ld1rLec2were studied in Marsupenaeus japonicus. LSm14A belongs to the LSm family and also plays an important role in the antiviral innate immunity. In this study, we mainly focus on the function of three newly identified MjLSm in PAMPs recognition in the antiviral innate immunity.1. Functional study of LdlrLecl and Ld1rLec2from shrimp Marsupenaeus japonicusC-type lectins belong to a large superfamily of carbohydrate recognition proteins that can bind to sugar moieties with single or multiple carbohydrate recognition domains (CRDs). CTLs are widely distribucted in living organisms. Lots of CTLs have been identified in crustacean. Most of them have only one CRD and are mainly expressed in hemocytes and hepatopancreas. Most shrimp CTLs can agglutinate bacteria in the presence of calcium. Although some of the CTLs have lost the ability of agglutination, they still play important roles in host immune defence as PRRs. Some CTLs can enhance hemocytes encapsulation, while some can kill bacteria directly besides their recognition ability. CTLs also play important roles in antiviral innate immune. Therefore, to study the antipathogenic mechanism underlying the antiviral response in shrimp plays an important role in understanding the host defence of pathogenic microorganisms and exploring effective immune prevention.The two CTLs expressed in all the tested tissues of shrimp, however, LdlrLec1was mainly expressed in hemocytes, heart, gill and intestines, whereas LdlrLec2was expressed in hepatopancreas and heart. The expression of both LdlrLecl and LdlrLc2 mRNA were obviously upregulated upon WSSV challenge. Injection of recombinant LdlrLecl and LdlrLec2into shrimp inhibited WSSV replication, and the accumulative mortalities were reduced while the shrimp were infected by WSSV. Knocking down the expression of LdlrLecl and LdlrLec2by RNA interference increased WSSV replication in vivo. The LdlrLec proteins could interact with VP28, a major envelope protein of WSSV, which is necessary for the attachment and penetration of WSSV into shrimp cells. The infection rates of WSSV incubated with LdlrLecs were reduced significantly compared with the control group. These results indicate that LdlrLec1and Ld1rLec2could bind with VP28through their CRD domain and inhibit the pervasion and replication of WSSV in shrimp, and play important roles in antiviral response.2. Functional study of MjLSm from shrimp Marsupenaeus japonicusLSm proteins are a family of RNA-binding proteins found in virtually every cellular organism. LSm proteins are defined by their assembly into rings of six or seven individual LSm protein molecules. The LSm complex plays an important role in pre-mRNA splicing and mRNA decapping. LSm14A, also named RAP55(mRNA-associated protein55), was defined as a new kind of PRRs and localizes to P-body. Human LSm14A can bind with virus nucleartide and translocate to peroxisomes and activate downstream signal pathway to mediate the induction of IFN-P in the early phase of viral infection and plays an important role in antiviral innate immune. But the function of LSm14A in crustacean has not been studied yet. In this study, three new LSm14As, MjhSm1, MjLSm2and MjLSm3were identified and the function and antivial mechanism were studied in Marsupenaeus japonicus.MjLSm1, MjLSm2and MjLSm3were widely distribucted in different tissues of shrimp. MjLSm1was mainly distribucted in hemocytes, heart and hepatopancreas, with a lower expression in gill. MjLSm2was mainly distribucted in hemocytes and hepatopancreas, with a lower expression in gill and intestine. MjLSm3was mainly distribucted in hepatopancreas, with a lower expression in heart and hemocytes, and very little in stomach and intestine. The proteins of all the three MjLSms, MjLSm1, MjLSm2and MjLSm3, contained a Sm domain at its N-terminal region and a FFD domain at its C-terminus. And all of the MjLSms were upregulated by WSSV challenge in gill. Gel-shift assay shows that MjLSml, MjLSm2and MjLSm3could bind with WSSV dsRNA. RNAi MjLSm promoted WSSV replication in early phase of WSSV infection in vivo and inhibited the expression of crustin4and crustinll. Overexpression of MjLSm1, MjLSm2and MjLSm3inhibited WSSV replication. The expression of crustin4was upregulated when infected with WSSV while MjLSm1or MjLSm2was overexpresed. The expression of crustinll was upregulated when infected with WSSV while MjLSml, MjLSm2or MjLSm3was overexpresed. These results indicate that MjLSm1, MjLSm2and MjLSm3play important roles in shrimp antivial innate immunity.