PmVRP15, a Novel Viral Responsive Protein from the Black Tiger Shrimp, Penaeus monodon, Promoted White Spot Syndrome Virus Replication

Abstract

Suppression subtractive hybridization of Penaeus monodon hemocytes challenged with white spot syndrome virus (WSSV) has identified the viral responsive gene, PmVRP15, as the highest up-regulated gene ever reported in shrimps. Expression analysis by quantitative real time RT-PCR revealed 9410–fold up-regulated level at 48 h post WSSV injection. Tissue distribution analysis showed that PmVRP15 transcript was mainly expressed in the hemocytes of shrimp. The full-length cDNA of PmVRP15 transcript was obtained and showed no significant similarity to any known gene in the GenBank database. The predicted open reading frame of PmVRP15 encodes for a deduced 137 amino acid protein containing a putative transmembrane helix. Immunofluorescent localization of the PmVRP15 protein revealed it accumulated around the nuclear membrane in all three types of shrimp hemocytes and that the protein was highly up-regulated in WSSV-infected shrimps. Double-stranded RNA interference-mediated gene silencing of PmVRP15 in P. monodon significantly decreased WSSV propagation compared to the control shrimps (injected with GFP dsRNA). The significant decrease in cumulative mortality rate of WSSV-infected shrimp following PmVRP15 knockdown was observed. These results suggest that PmVRP15 is likely to be a nuclear membrane protein and that it acts as a part of WSSV propagation pathway.

Limited evidence for white spot syndrome virus susceptibility associated with expression of PmVRP15 in local population of giant tiger shrimp (Penaeus monodon)

“…However, both the up-regulation of PmVRP15 in G 8 (P = 0.399) and down-regulation in G 8iA (P = 0.393) were not statistically significant (Fig. 3), contradicting a previous study that showed that PmVRP15 expression in the Thai population was significantly up-regulated in hemocytes at 48 hpi (Vatanavicharn et al, 2014). The disparity between this present study and Vatanavicharn et al (2014) can be explained in two ways.…”

“…Hemolymph was then poured into a microtube and kept on ice. The hemolymph (100 µl) sample was immediately centrifuged at 5,000 x g for 5 minutes at 4°C to separate the hemocyte (pellet) from the hemolymph (Vatanavicharn et al, 2014). The supernatant was discarded whereas the pellet (the hemocyte) was used in RNA isolation.…”

“…[forward] and 5' ACAGCGACTCCAAGGTCT ACGA 3' [reverse]) used in this study were based on those described in Vatanavicharn et al (2014). Thermal cycling was performed at 95°C for 3 minutes, 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 45 seconds.…”

“…PmVRP15 has been found as a nuclear membrane-like protein acting to mediate replication of WSSV in hemocytes in early infection (Vatanavicharn et al, 2014). A linear form of ∆C T (2 -∆C T ) showed that baseline expression (non-infected) of PmVRP15 in G 8iA was higher than G 8 .…”

“…A Penaeus monodon viral responsive protein, PmVRP15, has been found as a gene that is responsive to the infection of WSSV in 99 Al Haq et al hemocytes by mediating WSSV propagation at the acute phase (Vatanavicharn et al, 2014). Vatanavicharn et al (2014) also conducted RNA interference to alter PmVRP15 expression and elucidated that PmVRP15 was associated with WSSV susceptibility in a P. monodon population in Thailand. This signifies the importance of determining the change in expression of PmVRP15 in association with susceptibility to WSSV infection.…”