Identification and characterization of novel virus-responsive genes in the black tiger shrimp Penaeus monodon

Abstract

White spot syndrome virus (WSSV) and yellow head virus (YHV) are major viral pathogens for various crustaceans which cause severe losses to the shrimp aquaculture. Here, suppression subtractive hybridization (SSH) was employed to identify genes up-regulated in the hemocytes of WSSV- and YHV-infected Penaeus monodon at the early (24 h) and late phases (48 and 72 h) of the viral infections. A number of novel viral responsive genes regarding antiviral immunity and/or viral infection mechanism were uncovered. Among the viral responsive genes obtained from the SSH libraries, two novel genes/proteins first identified and named here, hemocyte homeostasis-associated protein (PmHHAP) and viral responsive protein 15 kDa (VRP15), showed to be greatly abundant in the SSH libraries. Hence, PmHHAP and VRP15 were further characterized for their roles in the shrimp’s response to viral infection. The full-length cDNA of PmHHAP showed high sequence identity to hypothetical proteins from various organisms, with the highest identity (54%) to the hypothetical protein TcasGA2_TC006773 from the red flour beetle, Tribolium castaneum. Transcripts of PmHHAP were expressed in various shrimp tissues with the highest expression in hematopoietic tissue. Upon WSSV infection, a high up-regulation level of shrimp hemocytic HHAP mRNA and protein was observed. Gene silencing of PmHHAP by RNA interference (RNAi) resulted in a significant decrease in the number of circulating hemocytes and 100% shrimp mortality within 30 h of the double-stranded PmHHAP RNA injection (but not in control shrimp), indicating that PmHHAP is essential for shrimp survival. Interestingly, severe damage of hemocytes was observed in vivo in the PmHHAP knockdown shrimp and in vitro in shrimp primary hemocyte cell culture, suggesting that PmHHAP plays an important role in hemocyte homeostasis. Thus, it is speculated that the upregulation of PmHHAP is an important mechanism to control circulating hemocyte levels in crustaceans during viral infection. The full sequence of VRP15 was obtained and showed no significant similarity (E-values > 1x10-4) to any known gene in the GenBank database. A protein domain prediction indicated that its protein structure consisted of a transmembrane helix. VRP15 transcript was mainly expressed in the hemocytes of shrimp, and found to be greatly up-regulated post WSSV-infection. Importantly, the significant reduction of WSSV replication was observed in the VRP15-RNAi knockdown shrimp, indicating that the novel VRP15 is important for viral propagation as its knock-down leads to decrease in viral infection in P. monodon.