Immune responses of whiteleg shrimp, Litopenaeus vannamei (Boone, 1931), to bacterially expressed dsRNA specific to VP28 gene of white spot syndrome virus.

In this study, dsRNA specific to VP28 gene of white spot syndrome virus (WSSV) of shrimp was synthesized in Escherichia coli in large scale and studied the immune response of shrimp to dsRNA-VP28. The haematological parameters such as clotting time and total haemocytes counts, and immunological parameters such as prophenoloxidase (proPO), superoxide dismutase (SOD), superoxide anion (SOA) and malondialdehyde content, as well as the mRNA expression of ten immune-related genes were examined to estimate the effect of dsRNA-VP28 on the innate immunity of Litopenaeus vannamei. The activities of proPO, SOA and SOD significantly increased in haemocyte after dsRNA-VP28 treatment, whereas MDA content did not change significantly. Among the ten immune-related genes examined, only the mRNA expression of proPO, cMnSOD, haemocyanin, crustin, BGBP, lipopolysaccharides (LPs), lectin and lysozyme in haemocytes, gill and hepatopancreas of L. vannamei, was significantly upregulated at 12 h after dsRNA-VP28 treatment, while no significant expression changes were observed in Toll receptor and tumour receptor genes. The increase of proPO and SOD activities, and SOA level and mRNA expression level of proPO, cMnSOD, haemocyanin, crustin, BGBP, LPs, lectin and lysozyme after dsRNA-VP28 stimulation indicate that these immune-related genes were involved in dsRNA-VP28-induced innate immunity in shrimp.

Taju G ，Madan N ，Abdul Majeed S ，Kumar TR ，Thamizhvanan S ，Otta SK ，Sahul Hameed AS ... -  《-》

Ontogenetic changes in the expression of immune related genes in response to immunostimulants and resistance against white spot syndrome virus in Litopenaeus vannamei.

In recent years, researchers have focused on viral and plant immunostimulants which could have beneficial effects in disease prevention and control in shrimp culture. At present, the application of the recombinant VP28 protein (r-VP28) and herbal immunostimulant has been considered as a more effective approach to prevent white spot syndrome (WSS) by enhancing the immune response in shrimp. In the present study, expression of selected immune related genes in response to r-VP28 and herbal immunostimulant mix (HIM) were separately studied qualitatively and quantitatively by RT-PCR and real time PCR, respectively during ontogenetic development from nauplius to juvenile stage in Litopenaeus vannamei. The mRNA expression level of immune related genes such as anti-lipopolysaccharides (ALF), Lysozyme, cMnSOD, Crustin, Prophenoloxidase, Tumor necrosis factor receptor-associated factor 6 (TRAF6) and Haemocyanin were found to be up-regulated significantly in different ontogenetic development stages of shrimp fed with r-VP28 and HIM formulated diets. Relative percent survival (RPS) was determined in shrimp fed with immunostimulants formulated diets after oral challenge with WSSV. The survival of WSSV challenged shrimp was found to be higher in immunostimulants treated groups when compared to untreated group. The results of PCR, ELISA and real time PCR revealed the absence of WSSV in WSSV-challenged shrimp after 20 days of treatment with immunostimulants. Among these immunostimulants, HIM was found to be more effective when compared to r-VP28. After a survey of literature, we are of the opinion that this might be the first report on the expression of immune genes during ontogenetic development of L. vannamei in response to immunostimulants.

Rajkumar T ，Taju G ，Abdul Majeed S ，Sinwan Sajid M ，Santhosh Kumar S ，Sivakumar S ，Thamizhvanan S ，Vimal S ，Sahul Hameed AS ... -  《-》

The promotion of cytoskeleton integration and redox in the haemocyte of shrimp Litopenaeus vannamei after the successive stimulation of recombinant VP28.

VP28 protein has been reported to work as a "vaccine" to protect the host from white spot syndrome, but the detailed mechanism of vaccination with VP28 protein in shrimp is still far from well understood. In the present study, whole transcriptomes of shrimp haemocytes were sequenced using the SOLiD4 platform after the successive VP28 stimulation. Eight single-end fragment libraries were constructed and sequenced in the four groups including the VP28-VP28, PBS-VP28, PBS-PBS and BLANK group, and there were 243,949,667 single-end reads with length of 50bp obtained totally, with 14,800 genes further identified. After reads mapping and transcript assembling, 1027, 1539, 1158, 1091 and 1300 genes in five differentially expressed gene lists were obtained in the comparison of VP28-VP28 versus PBS-VP28, VP28-VP28 versus PBS-PBS, VP28-VP28 versus BLANK, PBS-VP28 versus PBS-PBS and PBS-VP28 versus BLANK, respectively. There were 555 differentially expressed genes responsive to the single VP28 stimulation after grouping the PBS-VP28_BLANK and PBS-VP28_PBS-PBS gene lists, and 269 ones responsive to the successive VP28 stimulation after grouping the VP28-VP28_BLANK, VP28-VP28_PBS-PBS and VP28-VP28_PBS-VP28 gene lists. In the GO enrichment analysis of the genes responsive to the single VP28 stimulation, five immune-related GO terms were observed among 14 increased terms, which included defense response to bacterium, response to stimulus, disruption of cells of other organism, killing of cells of other organism and response to bacterium. It was worth noting that the GO terms, response to stimulus and response to stress, were the most common annotation ones which accounted 28.7% and 18.8% of the total differently expressed genes, respectively. For the genes responsive to the successive VP28 stimulation, terms including actin filament-based movement and myosin heavy chain binding were mostly enriched in the Biological Process and Molecular Function category, respectively. In the Cellular Component category, the enriched GO terms were myosin VII complex, myosin V complex, myosin VI complex and myosin II complex. Furthermore, the most abundant GO term was oxidation-reduction process, followed by single-organism transport, neurogenesis and translation for 214 genes only responsive to successive VP28 stimulation. These results collectively indicated that the successive VP28 stimulation could modulate cytoskeleton integration and redox to promote the phagocytosis activity of shrimp haemocytes, which might protect effectively for shrimp against WSSV infection.

Wang L ，Sun X ，Zhou Z ，Zhang T ，Yi Q ，Liu R ，Wang M ，Song L ... -  《-》

Bacillus subtilis spores expressing the VP28 antigen: a potential oral treatment to protect Litopenaeus vannamei against white spot syndrome.

The envelope protein VP28 of white spot syndrome virus (WSSV) is considered a candidate antigen for use in a potential vaccine to this important shrimp pathogen (the cause of white spot syndrome, WSS). Here, we used spores of Bacillus subtilis to display VP28 on the spore surface. Trials were conducted to evaluate their ability to protect shrimps against WSSV infection. The gene cotB-vp28 was integrated into the chromosome of the laboratory strain B. subtilis PY79, and expression of CotB-VP28 was detected by Western blotting and immunofluorescence. Expression of CotB-VP28 was equivalent to 1000 molecules per spore. PY79 and CotB-VP28 spores were mixed with pellets for feeding of whiteleg shrimps (Litopenaeus vannamei), followed by WSSV challenge. Superoxidase dismutase (SOD), phenoloxidase activities and mortality rates of the two shrimp groups were evaluated. Groups fed with PY79 and CotB-VP28 spores at day 7 had increased SOD activities of 29% and increased phenoloxidase activities of 15% and 33%, respectively, compared to those of the control group. Fourteen days postchallenge, 35% of vaccinated shrimps had died compared to 49% of those fed naked spores (PY79) and 66% untreated, unchallenged animals. These data suggest that spores expressing VP28 have potential as a prophylactic treatment of WSS.

Nguyen AT ，Pham CK ，Pham HT ，Pham HL ，Nguyen AH ，Dang LT ，Huynh HA ，Cutting SM ，Phan TN ... -  《-》

Injected phage-displayed-VP28 vaccine reduces shrimp Litopenaeus vannamei mortality by white spot syndrome virus infection.

White spot syndrome virus (WSSV) is the most important viral pathogen for the global shrimp industry causing mass mortalities with huge economic losses. Recombinant phages are capable of expressing foreign peptides on viral coat surface and act as antigenic peptide carriers bearing a phage-displayed vaccine. In this study, the full-length VP28 protein of WSSV, widely known as potential vaccine against infection in shrimp, was successfully cloned and expressed on M13 filamentous phage. The functionality and efficacy of this vaccine immunogen was demonstrated through immunoassay and in vivo challenge studies. In ELISA assay phage-displayed VP28 was bind to Litopenaeus vannamei immobilized hemocyte in contrast to wild-type M13 phage. Shrimps were injected with 2 × 10(10) cfu animal(-1) single dose of VP28-M13 and M13 once and 48 h later intramuscularly challenged with WSSV to test the efficacy of the vaccine against the infection. All dead challenged shrimps were PCR WSSV-positive. The accumulative mortality of the vaccinated and challenged shrimp groups was significantly lower (36.67%) than the unvaccinated group (66.67%). Individual phenoloxidase and superoxide dismutase activity was assayed on 8 and 48 h post-vaccination. No significant difference was found in those immunological parameters among groups at any sampled time evaluated. For the first time, phage display technology was used to express a recombinant vaccine for shrimp. The highest percentage of relative survival in vaccinated shrimp (RPS = 44.99%) suggest that the recombinant phage can be used successfully to display and deliver VP28 for farmed marine crustaceans.

Solís-Lucero G ，Manoutcharian K ，Hernández-López J ，Ascencio F ... -  《-》