Identification of a CqCaspase gene with antiviral activity from red claw crayfish Cherax quadricarinatus.

Caspase, an aspartate specific proteinase mediating apoptosis, plays a key role in immune response. In our previous study, the expression of a caspase gene was up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To further reveal the effect of caspase on WSSV infection, we cloned this caspase gene (denominated as CqCaspase) with an open reading frame of 1062 bp, which encoded 353 amino acids with a caspase domain (CASc) containing a p20 subunit and a p10 subunit. Tissue distribution analysis indicated that the mRNA transcript of CqCaspase was widely expressed in all tested tissues with the highest expression in Hpt, while the lowest expression in muscle. To further explore the effect of CqCaspase on WSSV replication, recombinant protein of CqCaspase (rCqCaspase) was delivered into Hpt cells followed by WSSV infection, which resulted in a significantly decreased expression of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV, suggesting that CqCaspase, possibly by the enhanced apoptotic activity, had a strong negative effect on the WSSV replication. These data together indicated that CqCaspase was likely to play a vital role in immune defense against WSSV infection in a crustacean C. quadricarinatus, which shed a new light on the mechanism study of WSSV infection in crustaceans.

A CqFerritin protein inhibits white spot syndrome virus infection via regulating iron ions in red claw crayfish Cherax quadricarinatus.

It is well known that iron is an essential element for all living organism. The intracellular iron availability is also important for the host's innate immune response to various pathogens, in which the iron homeostasis can be regulated by ferritin due to its iron storage property. In this study, a full-length cDNA sequence of ferritin (named as CqFerritin) was identified with 1410 bp from red claw crayfish Cherax quadricarinatus, which contained an open reading frame of 513 bp, encoding 170 amino acids with a conserved ferritin domain. Tissue distribution analysis demonstrated that CqFerritin was widely expressed in various tissues with high presence in haemocyte, haematopoietic tissue (Hpt) and heart, while lowest expression in hepatopancreas. In addition, loss-of-function of CqFerritin by gene silencing resulted in significantly higher expression of an envelope protein VP28 of white spot syndrome virus (WSSV) in red claw crayfish Hpt cell cultures, indicating the potential antiviral response of CqFerritin. To further explore the effect on WSSV replication by CqFerritin, recombinant CqFerritin protein (rCqFerritin) was transfected into Hpt cells followed by WSSV infection. Importantly, the replication of WSSV was obviously decreased in Hpt cells if transfected with rCqFerritin protein, suggesting that CqFerritin had clearly negative effect on WSSV infection. Furthermore, intracellular accumulation of iron ions was found to promote the WSSV replication in a dose-dependent manner, illustrating that the iron level regulated by CqFerritin was likely to be vital for WSSV infection in red claw crayfish. Taken together, these data suggest that CqFerritin plays an important role in immune defense against WSSV infection in a crustacean C. quadricarinatus.

CqToll participates in antiviral response against white spot syndrome virus via induction of anti-lipopolysaccharide factor in red claw crayfish Cherax quadricarinatus.

It is well known that Tolls/Toll like receptors (TLRs), a family of pattern recognition receptors, play important roles in immune responses. Previously, we found that a Toll transcript was increased in a transcriptome library of haematopoietic tissue (Hpt) cells from the red claw crayfish Cherax quadricarinatus post white spot syndrome virus infection. In the present study, a full-length cDNA sequence of Toll receptor (named as CqToll) was identified with 3482 bp which contained an open reading frame of 3021 bp encoding 1006 amino acids. The predicted structure of CqToll protein was composed of three domains, including an extracellular domain of 19 leucine-rich repeats residues, a transmembrane domain and an intracellular domain of 138 amino acids. Tissue distribution analysis revealed that CqToll was expressed widely in various tissues determined from red claw crayfish with highest expression in haemocyte but lowest expression in eyestalk. Importantly, significant lower expression of the anti-lipopolysacchride factor (CqALF), an antiviral antimicrobial peptide (AMP) in crustaceans, but not CqCrustin was observed after gene silencing of CqToll in crayfish Hpt cell cultures, indicating that the CqALF was likely to be positively regulated via Toll pathway in red claw crayfish. Furthermore, the transcription of both an immediate early gene and a late envelope protein gene VP28 of WSSV were clearly enhanced in Hpt cells if silenced with CqToll, suggesting that the increase of WSSV replication was likely to be caused by the lower expression of the CqALF resulted from the loss-of-function of CqToll. Taken together, these data implied that CqToll might play a key role in anti-WSSV response via induction of CqALF in a crustacean C. quadricarinatus.