Antiviral properties of Penaeus monodon cyclophilin A in response to white spot syndrome virus infection in the black tiger shrimp.

Cyclophilin A (CypA) or peptidylprolyl isomerase A, plays an important role in protein folding, trafficking, environmental stress, cell signaling and apoptosis etc. In shrimp, the mRNA expression level of PmCypA was stimulated by LPS. In this study, all three types of shrimp hemocytes: hyaline cell, granulocyte and semi-granulocyte expressed the PmCypA protein. The mRNA expression level of PmCypA was found to be up-regulate to four-fold in white spot syndrome virus (WSSV) infected hemocytes at 48 h. Interestingly, PmCypA protein was only detected extracellularly in shrimp plasma at 24 h post WSSV infection. To find out the function of extracellular PmCypA, the recombinant PmCypA (rPmCypA) was produced and administrated in shrimp primary hemocyte cell culture to observe the antiviral properties. In rPmCypA-administrated hemocyte cell culture, the mRNA transcripts of WSSV intermediate early gene, ie1 and early gene, wsv477 were significantly decreased but not that of late gene, vp28. To explore the antiviral mechanism of PmCypA, the expression of PmCypA in shrimp hemocytes was silenced and the expression of immune-related genes were investigated. Surprisingly, the suppression of PmCypA affected other gene expression, decreasing of penaeidin, PmHHAP and PmCaspase and increasing of C-type lectin. Our results suggested that the PmCypA might plays important role in anti-WSSV via apoptosis pathway. Further studies of PmCypA underlying antiviral mechanism are underway to show its biological function in shrimp immunity.

Plasmolipin, PmPLP1, from Penaeus monodon is a potential receptor for yellow head virus infection.

Plasmolipin has been characterized as a cell entry receptor for mouse endogenous retrovirus. In black tiger shrimp, two isoforms of plasmolipin genes, PmPLP1 and PmPLP2, have been identified from the Penaeus monodon EST database. The PmPLP1 is highly up-regulated in yellow head virus (YHV)-infected shrimp. Herein, the function of PmPLP1 is shown to be involved in YHV infection. The immunoblotting and immunolocalization showed that the PmPLP1 protein was highly expressed and located at the plasma membrane of gills from YHV-infected shrimp. Moreover, the PmPLP1 expressed in the Sf9 insect cells resided at the cell membrane rendering the cells more susceptible to YHV infection. Using the ELISA binding and mortality assays, the synthetic external loop of PmPLP1 was shown to bind the purified YHV and neutralize the virus resulting in the decrease in YHV infection. Our results suggested that the PmPLP1 was likely a receptor of YHV in shrimp.

Alpha-2-macroglobulin is a modulator of prophenoloxidase system in pacific white shrimp Litopenaeus vannamai.

The shrimp multifunctional protein alpha-2-macroglobulin (A2M) is abundantly expressed in plasma, highly up-regulated upon microbial infection and involved in several immune pathways such as blood clotting system, phagocytosis and melanization. Herein, the function of LvA2M from Litopenaeus vannamei on the prophenoloxidase (proPO) system is reported. The recombinant (r)LvA2M produced strongly and specifically inhibited trypsin and the PO activity in shrimp plasma in a dose-dependent manner. Silencing of LvA2M led to an increase in the PO activity in shrimp plasma although the expression of proPO-associated genes, proPO-activating enzyme (PPAE) and prophenoloxidase (proPO) but not the proPO-activating factor (PPAF) was down-regulated. In Vibrio parahaemolyticus AHPND-infected shrimp, the LvA2M activity was suppressed in an early phase of infection while the PO activity was increased. Thus, the proPO-activating system was regulated by the LvA2M.

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.

Two plasmolipins from the black tiger shrimp, Penaeus monodon and their response to virus pathogens.

Two isoforms of plasmolipin were initially identified from the black tiger shrimp (Penaeus monodon) EST database and completed using 50 RACE to reveal complete cDNAs of 558 bp (PmPLP1) and 537 bp(PmPLP2) with 87% nucleotide sequence identity. The deduced amino acid sequences contained four-transmembrane domains and showed the highest amino acid identity (49% and 51%, respectively) to the honey bee (Apis mellifera) chemokine-like factor (CKLF), with a very similar hydrophobic pattern to other plasmolipins. Transcripts of PmPLP1 and PmPLP2 were observed in all tested shrimp tissues with the highest expression levels in the gill and epipodite for PmPLP1 and in the hemocytes and antennal gland for PmPLP2. PmPLP1 transcript levels were significantly upregulated in hemocytes at 24 and 72 h post infection (hpi) with yellow head virus (YHV) (7.4- and 14.7- fold, respectively), but only after 72 hpi by white spot syndrome virus (WSSV). In contrast, PmPLP2 was only slightly (but statistically significant)up-regulated with YHV and WSSV. Thus, PmPLPs have the potential to be a part of viral infection mechanisms or defense response. This is the first characterization of a plasmolipin gene in crustaceans.

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.

Genomic structure, expression pattern and functional characterization of crustinPm5, a unique isoform of crustin from Penaeus monodon.

A unique isoform of crustin, crustinPm5, was identified from a gill-epipodite cDNA library of the tiger shrimp, Penaeus monodon. The crustinPm5 cDNA contains an open reading frame (ORF) of 510 bp encoding a 169 amino acid protein. The deduced amino acid sequence of crustinPm5 showed 38% and 37% overall sequence identity with those of crustinPm1 and crustin-likePm, respectively, two crustin isoforms previously reported. The crustinPm5 gene contained four exons interrupted by three introns whilst the upstream sequence contains a putative promoter with two potential binding sites for NF-kappaB, one complete heat-shock regulatory element (HSE) and five putative GATA factor binding sites. The transcripts of crustinPm5 were primarily observed in the epipodite and eyestalk and not in hemocytes. Expression analysis revealed that the transcript levels of crustinPm5, crustinPm1 and crustin-likePm in epipodite were up-regulated upon heat treatment and hyperosmotic salinity stress. The recombinant crustinPm5 exhibited antimicrobial activity against some Gram-positive bacteria in vitro, but did not inhibit the growth of any Gram-negative bacteria tested. These results, together with the transcript expression pattern, indicate a diverse function of the proteins in the crustin family particularly crustinPm5 that might function as a stress mediator in addition to its antibacterial action.