Abstract
Anti-lipopolysaccharide factors (ALFs) exhibit a potent antimicrobial activity against a broad range of bacteria, filamentous fungi, and viruses. In previous reports, seven groups of ALFs (groups A–G) were identified in penaeid shrimp. Among them, group D showed negative net charges and weak antimicrobial activity. Whether this group has antiviral function is not clear. In this study, the ALF sequences of penaeid shrimp were analyzed, and eight groups of ALF family (groups A–H) were identified. The four ALFs including MjALF-C2, MjALF-D1, MjALF-D2, and MjALF-E2 from kuruma shrimp Marsupenaeus japonicus were expressed recombinantly in Escherichia coli, and the antiviral activity was screened via injection of purified recombinant ALFs into shrimp following white spot syndrome virus (WSSV) infection. Results showed that the expression of Vp28 (WSSV envelope protein) decreased significantly in the MjALF-D2-injected shrimp only. Therefore, MjALF-D2 was chosen for further study. Expression pattern analysis showed that MjAlf-D2 was upregulated in shrimp challenged by WSSV. The WSSV replication was detected in RNA, genomic DNA, and protein levels using VP28 and Ie1 (immediate-early gene of WSSV) as indicators in MjALF-D2-injected shrimp following WSSV infection. Results showed that WSSV replication was significantly inhibited compared with that in the rTRX- or PBS-injected control groups. After knockdown of MjAlf-D2 in shrimp by RNA interference, the WSSV replication increased significantly in the shrimp. All these results suggested that MjALF-D2 has an antiviral function in shrimp immunity, and the recombinant ALF-D2 has a potential application for viral disease control in shrimp aquaculture.
Highlights
White spot syndrome (WSS) in crustaceans is caused by WSS virus (WSSV), which is an enveloped circular doublestranded DNA virus, and it is a severely infectious disease that results in 100% mortality of shrimp within 3–10 days after infection (Peng et al 2001)
29 sequences of Anti-lipopolysaccharide factors (ALFs) (GenBank accession numbers listed in Fig. 1A) were collected in four other penaeid shrimp species, namely, F. chinensis, F. penicillatus, L. vannamei, and P. monodon, from the GenBank database for reconstruction of phylogenetic trees
Eight groups of ALF family were identified in kuruma shrimp M. japonicus, and there has not been any functional study conducted for group D member MjALF-D2
Summary
White spot syndrome (WSS) in crustaceans is caused by WSS virus (WSSV), which is an enveloped circular doublestranded DNA virus, and it is a severely infectious disease that results in 100% mortality of shrimp within 3–10 days after infection (Peng et al 2001). WSSV is a virulent pathogen to shrimp and to wider crustacean species. Several kinds of AMPs have been identified in shrimp, including penaeidins, crustins, anti-lipopolysaccharide factors (ALFs), and stylicins (Tassanakajon et al 2011, 2018). ALF was first reported in chelicerates (horseshoe crab, Limulus polyphemus) as a potent anticoagulant factor (Morita et al 1985; Ohashi et al 1984; Tanaka et al 1982). ALFs were identified in various crustaceans, such as portunid crabs (Afsal et al 2012), freshwater prawn (Ren et al 2012), crayfish
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