Antilipopolysaccharide Factor Interferes with White Spot Syndrome Virus Replication In Vitro and In Vivo in the Crayfish Pacifastacus leniusculus

Abstract

In a study of genes expressed differentially in the freshwater crayfish Pacifastacus leniusculus infected experimentally with the white spot syndrome virus (WSSV), one protein, known as antilipopolysaccharide factor (ALF), was chosen, among those whose transcript levels increased upon viral infection, for further studies. ALF RNA interference (RNAi) experiments in whole animals and in cell cultures indicated that ALF can protect against WSSV infection, since knockdown of ALF by RNAi specifically resulted in higher rates of viral propagation. In a cell culture of hematopoietic tissue (Hpt) from P. leniusculus, quantitative PCR showed that knockdown of ALF by RNAi resulted into WSSV levels that were about 10-fold higher than those treated with control double-stranded RNA (dsRNA). In addition, RNAi experiments with other crayfish genes that had been found to be up-regulated by a WSSV infection did not result in any changes of viral loads. Thus, the cell culture does not respond to dsRNA in a similar manner, as shown earlier for dsRNA injected into shrimp, which gave a higher degree of resistance to WSSV infection. If ALF transcription in whole animals was stimulated by the administration of UV-treated WSSV, a partial protection against a subsequent challenge with the active virus was conferred to the host. This is the first crustacean gene product identified with the capacity to interfere with replication of this important pathogen.

Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis

“…Considering the new finding that ALF could interfere with WSSV propagation both in vitro and in vivo in the crayfish P. leniusculus [23], EsALF provided a candidate promising therapeutic or prophylactic agent in aquaculture.…”

“…Subsequent studies indicated that ALF from L. polyphemus exhibited strong antibacterial activity against Gram-negative R-type bacteria through neutralizing the lipid A, a common toxic moiety of LPS [21,22], and also the inhibitory effect on the growth of Gram-positive bacteria. Afterward, similar proteins were identified from the haemocytes of Litopenaeus vannamei [15], T. tridentatus [20], Penaeus monodon [17,18], Fenneropenaeus chinensis [9], Marsupenaeus japonicus [16], Pacifastacus leniusculus [23], and Scylla paramamosain [24], etc. The expression profile of ALFs indicated that the mRNA transcripts for the protein could be induced by Vibrio infection [9,18] or LPS administration [16], suggesting their crucial roles in crustacean innate immunity.…”

RNA Interference with Special Reference to Combating Viruses of Crustacea

“…This technique has been some what underexploited regarding crustaceans, perhaps due to the lack of a readily available in vitro cell culture system. dsRNA targeting crayfish anti-lipopolysaccharide factor (ALF) resulted in a tenfold increase in WSSV demonstrating a critical role of ALF in combating WSSV [112]. Target RNAi assays against P. monodon Rab 7 GTPase involved with viral endocytosis in endosome plus an anti-YHV resulted in 20 % less mortality than the anti-YHV alone [143].…”

“…Early studies showed down regulation of genes based on the presence/absence or lighter amplicons on gels [112,155,207]. When the titre was measured by quantitative PCR, the viral titre was reduced twofold to 1 log reduction.…”

The scope of the crustacean immune system for disease control

“…Crustin antimicrobial peptides have been reported to have a role in the immune response to infection with Gram-positive and Gram-negative bacteria, fungi and viruses (Table 1) Vega et al, 2008). Also, it has been reported from RNA inhibition (RNAi) studies of ALF transcription in Pacifastacus leniusculus haemopoeitic tissue cell culture that ALFs may act by directly interfering with viral replication (Liu et al, 2006). However, as discussed further below, the reported broad and perhaps conflicting spectrum of activity of different classes of antimicrobial peptide may, in part, be confounded by the use of in vitro methods in isolation to determine activity.…”