The anti-lipopolysaccharide factor (ALF) from the black tiger shrimp, Penaeus monodon, has been shown previously to exhibit a broad spectrum of activity against various strains of bacteria and fungi. Herein, the recombinant ALFPm3 (rALFPm3) protein was examined for its role in the defence against white spot syndrome virus (WSSV) infection in haematopoietic (Hpt) cell cultures of the freshwater crayfish, Pacifastacus leniusculus, as well as in live P. monodon shrimps. Incubation of Hpt cell cultures with a mixture of WSSV and rALFPm3 resulted in a dosedependent decrease in VP28 gene expression levels, compared with those incubated with WSSV alone, with an rALFPm3 IC 50 value lower than 2.5 mM. However, pre-treatment of Hpt cells with 5 mM rALFPm3 showed no induced protection against subsequent WSSV infection, whereas the synthetic crayfish ALF peptide could protect cells at a higher concentration (10 mM). The in vivo role of ALFPm3 was examined by injection of P. monodon with WSSV pre-treated with rALFPm3 protein. The results clearly showed that rALFPm3 was able to reduce WSSV propagation and prolong the survival of shrimps.
INTRODUCTIONWhite spot syndrome (WSS) is a severe infectious disease in shrimps as well as other crustaceans (Flegel, 1997;Lo et al., 1996), caused by white spot syndrome virus (WSSV), and causes great losses in commercial shrimp production. The mortality of WSSV-infected shrimp can reach 100 % within 3-10 days after infection. Attempts to protect or control the WSSV infection include vaccination of shrimps with viral envelope proteins, using RNA interference to induce viral immunity and direct neutralization by antiviral proteins (Liu et al., 2009;Luo et al., 2003;Robalino et al., 2007;Roch et al., 2008; Witteveldt et al., 2004a, b;Zhang et al., 2004).The existence of antiviral properties of several peptides has been reported in penaeid shrimps. PmAV protein, a c-type lectin from the black tiger shrimp, Penaeus monodon, strongly inhibits virus-induced cytopathic effects in a fish cell culture (Luo et al., 2003). Haemocyanin, a group of antibacterial and antifungal peptides in shrimps, also exhibits antiviral property against WSSV in the closely related Marsupenaeus japonicus (Lei et al., 2008).Moreover, haemocyanin from P. monodon was observed to have non-specific antiviral properties in fish cell cultures in the absence of detected cytotoxicity against the host cells (Zhang et al., 2004). More recently, the newly discovered ctype lectin from Litopenaeus vannamei was shown to exhibit antiviral activity against WSSV (Zhao et al., 2009). In molluscs, synthetic mytilin, an antibacterial peptide from Mytilus galloprovincialis, possesses in vitro anti-WSSV activity and reduces the mortality of WSSV-infected palaemonid shrimps (Dupuy et al., 2004;Roch et al., 2008).Anti-lipopolysaccharide factor (ALF), an antimicrobial protein, was first discovered in the horseshoe crabs, Tachypleus tridentatus (TALF) and Limulus polyphemus (LALF) (Aketagawa et al., 1986; Morita et al., 1985;Tanaka et al., 1982),...