PmVRP15, a novel viral responsive protein from the black tiger shrimp, Penaeus monodon, promoted white spot syndrome virus replication.

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.

A Kazal-type serine proteinase inhibitor SPIPm2 from the black tiger shrimp Penaeus monodon is involved in antiviral responses.

A five-domain Kazal-type serine proteinase inhibitor, SPIPm2, from Penaeus monodon has recently been implicated in antiviral responses for it is up-regulated upon viral infection and needs further studies. The SPIPm2 genomic gene was composed of seven exons and six introns. The genomic DNA segments coding for each Kazal domain were separated by introns of variable lengths supporting the hypothesis of gene duplication in the Kazal-type gene family. RT-PCR and Western blot analysis revealed that the SPIPm2 transcript and its five-domain protein product were expressed mainly in the hemocytes and less in gill, heart and antennal gland. Upon white spot syndrome virus (WSSV) infection, the SPIPm2 was only detected in the hemocytes and plasma. Immunocytochemical study of P. monodon hemocytes showed that the percentage of SPIPm2-producing hemocytes was reduced by about half after WSSV infection. Quantitative RT-PCR revealed further that the SPIPm2 was up-regulated early in the hemocytes of WSSV-infected shrimp and gradually reduced as the infection progressed. Injection of the recombinant SPIPm2 (rSPIPm2) prior to WSSV injection resulted in a significant inhibition of WSSV replication. The rSPIPm2 injection also prolonged the mortality rate of WSSV-infected shrimp. Therefore, the SPIPm2 was involved in the innate immunity against WSSV infection in shrimp.

A novel viral responsive protein is involved in hemocyte homeostasis in the black tiger shrimp, Penaeus monodon.

A novel viral responsive protein, namely hemocyte homeostasis-associated protein (HHAP), was characterized for its role in the response of shrimp to white spot syndrome virus infection. The full-length cDNAs of HHAP from the black tiger shrimp (PmHHAP), Penaeus monodon, and the fresh water crayfish (PlHHAP), Pacifastacus leniusculus, were obtained and showed high sequence identity to a hypothetical protein from various organisms, with the highest identity to the hypothetical protein TcasGA2_TC006773 from the red flour beetle, Tribolium castaneum (54% amino acid sequence identity). Transcripts of PmHHAP were expressed in various shrimp tissues with the highest expression in hematopoietic tissue, whereas the transcripts of PlHHAP were found in the hematopoietic and nerve tissues. Upon white spot syndrome virus infection, a high up-regulation level of shrimp hemocytic HHAP mRNA and protein was observed by real-time reverse transcription-PCR and immunofluorescence microscopy, respectively. Gene silencing of PmHHAP by RNA interference 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 HHAP 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 up-regulation of PmHHAP is an important mechanism to control circulating hemocyte levels in crustaceans during viral infection.

Identification of genes expressed in response to yellow head virus infection in the black tiger shrimp, Penaeus monodon, by suppression subtractive hybridization.

Suppression subtractive hybridization (SSH) was employed to identify yellow head virus (YHV)-responsive genes from the hemocytes of the black tiger shrimp, Penaeus monodon. Two SSH cDNA libraries were constructed to identify viral responsive genes in the early (24I) and late (48/72I) phases of YHV infection. From 240 randomly selected clones from each library, 155 and 30 non-redundant transcripts were obtained for the early and late libraries, respectively. From these clones, 72 and 16, respectively, corresponded to known genes (E-values < 1 x 10(-4)) that could be categorized according to their putative functions. The upregulated genes identified as likely to be associated with cell defense and homeostasis were found at a high proportion in the 24I SSH library, but not in 48/72I SSH library implying that these immune molecules participate in viral defense immunity in the early phase of YHV infection whereas their expressions were suppressed in the late phase of infection. Novel YHV-responsive genes were uncovered from these SSH libraries including caspases, histidine triad nucleotide-binding protein 2, Rab11, beta-integrin, tetraspanin, prostaglandin E synthase, transglutaminase, Kazal-type serine proteinase inhibitor and antimicrobial peptides. Among these YHV-responsive genes, several have been previously reported to participate in defense against white-spot syndrome virus (WSSV) implying that YHV infection in shrimp induces similar host immune responses as observed during WSSV infection. The expression of four apparently upregulated immune-related genes identified from the two SSH libraries, anti-lipopolysaccharide factor isoform 6 (ALFPm6), crustin isoform 1 (crustinPm1), transglutaminase and Kazal-type serine proteinase inhibitor isoform 2 (SPIPm2), was evaluated by real-time RT-PCR to reveal differential expression in response to YHV infection at 6, 24, 48 and 72 h post-infection. The results confirmed their differential expression and upregulation, and thus verified the success of the SSHs and the likely involvement of these genes in shrimp antiviral mechanisms.