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Abstract: Peroxiredoxins (Prx) are a family of antioxidant proteins and perform important functions in intracellular signal transduction. Here, we report a Prx gene from Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA of FcPrx gene contained an open reading frame of 735 bp encoding a polypeptide of 275 amino acids. The molecular mass of the deduced amino acid of FcPrx is 27445.43 Da with an estimated pI of 5.71. Sequence comparison showed that the FcPrx shares high identities with Prx IVs and it was named FcPrx4. A real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of FcPrx4 in different tissues and temporal expression in hemocytes and hepatopancreas of F. chinensis challenged by white spot syndrome virus (WSSV). Transcripts of FcPrx4 can be detected in all tissues examined. The expression of FcPrx4 showed significant up-regulation in shrimp hemocytes and hepatopancreas after artificial infection with WSSV. A fusion protein containing FcPrx4 was produced in vitro and was confirmed by Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) assay. And activity analysis indicated that the recombinant FcPrx4 proteins can reduce H2O2 in the presence of dithiothreitol. [Copyright &y& Elsevier]

The specific binding between viral attachment proteins (VAPs) of a virus and its cellular receptors on host cells mediates virus entry into host cells, which triggers subsequent viral infections. Previous studies indicate that F 1 ATP synthase β subunit (named BP53), is found on the surface of shrimp cells and involved in white spot syndrome virus (WSSV) infection by functioning as a potential viral receptor. Herein, in a far-western blotting assay, three WSSV proteins with molecular weights of 28 kDa, 37 kDa, and >50 kDa were found to interact with BP53. The 28 kDa and 37 kDa proteins were identified as the envelope protein VP28 and VP37 of WSSV respectively, which could be recognized by the polyclonal antibodies. Enzyme-linked immunosorbent binding assays revealed that VP37 contributed to almost 80% of the binding capability for BP53 compared with the same amount of total WSSV protein. The relationship between BP53 and its complementary interacting protein, VP37, was visualized using a co-localization assay. Bound VP37 on the cell surface co-localized with BP53 and shared a similar subcellular location on the outer surface of shrimp cells. Pearson’s correlation coefficients reached to 0.67 ± 0.05 and the Mander’s overlap coefficients reached 0.70 ± 0.05, which indicated a strong relationship between the localization of BP53 and bound rVP37. This provides evidence for an interaction between BP53 and VP37 obtained at the molecular and cellular levels, supporting the hypothesis that BP53 serves as a receptor for WSSV by binding to VP37. The identification of the viral binding proteins of shrimp BP53 is helpful for better understanding the pathogenic mechanisms of WSSV to infect shrimp at the cellular level. [ABSTRACT FROM AUTHOR]

Abstract: To test the possibility that shrimp pond rotifer resting eggs and hatched rotifers could transmit white spot syndrome virus (WSSV) to crayfish (Procambarus clarkii), we injected crayfish with rotifer and resting egg inocula that were WSSV-positive only by dot-blot analysis of PCR products. No crayfish became WSSV-positive after challenge with the resting egg inoculum. However, 1/15 crayfish became WSSV-positive after challenge with the rotifer inoculum. The results demonstrated that rotifers constitute a potential risk for WSSV transmission to crayfish and other cultivated crustaceans. However, the actual quantitative risk of transmission in an aquaculture setting depends on many variables that remain untested. [Copyright &y& Elsevier]

WSSV is one of the most harmful pathogeny in the pacific white shrimp, and genetic variations caused the strains of different virulence. MicroRNAs (miRNAs) involved in the regulation of virus defense. To understand the different virulence of WSSV on miRNA expression in Litopeneaus vannamei , the deep sequencing was performed to compare two small RNA libraries prepared from hepatopancreas of Litopeneaus vannamei infected with normal-virulence or low-virulence WSSV. Approximately 29,398,623 raw reads from normal-virulence library and 35,291,803 raw reads from low-virulence library were obtained. There were about 37 miRNAs homologs identified. Sixteen miRNAs were significantly up-regulated and twenty-one miRNAs were significantly down-regulated in normal-virulence infection library compared with low-virulence infection library. Of these, Igi-miR-1175-3p was the most significant different miRNA, followed by bmo-miR-1175-3p and ipu-miR-26b, respectively. The putative target genes for differentially expressed miRNAs were concerned with biological processes, signal meditated, cell differentiation and apoptosis, immune recognition and other more functions. The results will help to understand the miRNAs response to different virulence WSSV infection. [ABSTRACT FROM AUTHOR]

This study conducted a 30-day feeding trial and a subsequent 20-day anti-virus infection trial to determine the effects of probiotic Bacillus PC465 on the growth, health status, and disease resistance of Litopenaeus vannamei . Shrimp samples were fed with three practical diets prepared from shrimp feed containing varying probiotic doses [0 (control), 10 7 , and 10 9 CFU g −1 ]. Probiotic supplementation significantly increased the weight gain and survival of L. vannamei ( p < 0.05). The effect of 10 9 CFU g −1 on the growth rate was higher than that of 10 7 CFU g −1 . Compared with those in the control group, the activities of digestive enzymes, such as amylase, protease, and lipase, in the shrimp mid-gut significantly increased in the probiotic-fed groups on days 15 and 30, except lipase on day 30. The influence of 10 9 CFU g −1 on enzyme activities was also greater than that of 10 7 CFU g −1 . Scanning electron microscopy revealed folds and large ravines across the interior surface of the mid-gut, and the number of these folds and ravines increased significantly after the probiotic was administered. The probiotic treatment significantly ( p < 0.05) enhanced the transcription of penaeidin 3 a (Pen-3 a ), peroxinectin, C-type lectin 3 (Lec-3), and thioredoxin (Trx) in the hemocytes of L. vannamei . Likewise, probiotic treatment increased the transcription of hemocyanin in the hepatopancreas of L. vannamei . The probiotic treatment also significantly increased the transcription of prophenoloxidase (proPO) but decreased the transcription of crustin in hemocytes. By contrast, the same treatment failed to increase the transcription of Ras-related protein (Rab-6) in hemocytes. The number of species and biomass of Bacillus in the mid-gut were higher in the probiotic-fed group than in the control group. The total biomass of microbes was higher in the shrimp fed with 10 7 CFU g −1 than in the shrimp fed with 10 9 CFU g −1 and the control group on days 15 and 30 post-feeding. In two white spot syndrome virus (WSSV) infections, the weight gain, survival, and WSSV copies within the gills of the probiotic-treated shrimp significantly differed ( p < 0.05) from those of the control group. Relatively efficient protection was associated with probiotic feeding. Results suggested that Bacillus PC465 feeding improves the growth performance, survival, digestion, and nutrient absorption of L. vannamei . Probiotic treatment also enhances the microbial structures in the gut, promotes the immune status of shrimp, and provides protection against viral infection. The supplementation with 10 9 CFU g −1 can also improve the growth and survival of L. vannamei . [ABSTRACT FROM AUTHOR]

White spot syndrome virus (WSSV) is the main pathogen of shrimp culture, and has brought great losses of the shrimp aquaculture industry every year since it has been found. However, the specific mechanism of the virus into the cell is not very clear. Recent research suggests that clathrin-mediated endocytosis is involved in WSSV infection. By sequence analysis, clathrin coat AP17 is an σ subunit of AP-2 complex which is involved in clathrin-mediated endocytosis. To obtain the full-length sequence of Clathrin coat AP17 of Litopenaeus vannamei ( LvCCAP17 ), the rapid amplification of cDNA ends (RACE) was performed to get the sequence of 3'and 5′ end and splicing by DNAMAN. The full-length sequence of LvCCAP17 is 842 bp and expected to encoding 142 amino acids, and the amino acid sequence was analyzed by online software. The mRNA expression of LvCCAP17 in different tissues was carried out with quantitative real-time PCR and the LvCCAP17 was detected in all tested tissues of Litopenaeus vannamei . The transcriptional expression level of LvCCAP17 in epithelium and hepatopancreas was significantly up-regulated after WSSV infection. Far-Western blotting and ELISA assay showed that LvCCAP17 interacted with rVP26 and rVP37. Silencing of LvCCAP17 gene by double-strand RNA (dsRNA) interference significantly delay of cumulative mortality rate in WSSV infected shrimp and reduced the expression level of immediate early gene 1 ( ie1 ) and vp28 . These results indicated that clathrin-meated endocytosis is responsible for WSSV infection. [ABSTRACT FROM AUTHOR]

Thioredoxin (TRX), a major intracellular antioxidant, has a wide range of biological functions. It was up-regulated and targeted by WSSV. However, the relevance of TRX with WSSV infection and signaling pathway remains largely unknown. Sequence analysis indicated that TRX might interact with the WSSV030 (VP362) and WSSV454 (thymidine kinase–thymidylate kinase, TK-TMK) of WSSV. In this study, TRX, VP362 and TK-TMK were expressed and the interaction of TRX with VP362 or TK-TMK was investigated. Furthermore, how TRX affect the process of WSSV infection and the gene transcription of inhibitor of nuclear factor kappa-B kinase (IKK), a p38 mitogen-activated protein kinase (LvP38) and signal transducer and activator of transcription (STAT) in the hemocytes and hepatopancreas was explored. Far-western blot and enzyme-linked immuno assay (ELISA) results showed that TRX interacted with VP362 and TK-TMK. The mRNA expressions of IKK, LvP38 and STAT were significantly affected by the over-presence of TRX of Litopenaeus vannamei . Neutralization experiment in vivo indicated that TRX induced the transcription expression of VP28 and increased the viral copy numbers in the early stage of WSSV infection and it may attribute to the death of shrimps infected by WSSV. [ABSTRACT FROM AUTHOR]

The interaction between viral structural proteins and host plays key functions in viral infection. In previous studies, most research have been undertaken to explore the interaction of envelope structural proteins with host molecules. However, how the nucleocapsid proteins of WSSV interacted with host molecules remained largely unknown. In this study, the interaction of nucleocapsid protein VP51 and ribosomal protein L7 of Litopenaeus vannamei (LvRPL7) was reported. Furthermore, the mRNA transcriptional response of LvRPL7 to WSSV was investigated. The results showed that LvRPL7 was widely distributed in all analyzed tissues of L. vannamei . The high expression levels of LvRPL7 were found in the tissues of muscle and gills. The temporal expression of LvRPL7 in WSSV-challenged shrimp showed that LvRPL7 was up-regulated (P < 0.5) in the muscle at 8 h and 24 h post WSSV challenge and then restored to the normal levels. But the LvRPL7 expression was up-regulated (P < 0.5) in the hepatopancreas at 8 h post WSSV challenge and down-regulated at 12 h and 24 h post WSSV challenge. Indirect immunofluorescence assay indicated that LvRPL7 was mainly located on the surface and cytoplasm of hemocytes. Far-Western blotting showed that VP51 bound with LvRPL7. Moreover, ELISA results appeared that LvRPL7 interacted with VP51 in concentration dependent manner. Neutralization assay in vivo showed that anti-LvRPL7 antibody significantly delayed WSSV infection. Our results reveal that LvRPL7 was involved in WSSV infection. [ABSTRACT FROM AUTHOR]

Cytokines play a critical role in innate and adaptive immunity. Astakines represent a group of invertebrate cytokines that are related to vertebrate prokineticin and function in promoting hematopoiesis in crustaceans. We have identified an astakine from the white shrimp Litopeneaus vannamei and named it LvAST in a previous research. In the present research, we investigated the interactions among LvAST, the envelope protein VP37 of white spot syndrome virus (i.e., WSSV), and the β subunit of F 1 -ATP synthase (ATPsyn-β) of the white shrimp (i.e., BP53) using binding assays and co-precipitations. We also examined the effects of LvAST on shrimp susceptibility to WSSV. We found that LvAST and VP37 competitively bound to BP53, but did not bind to each other. Shrimps that had been injected with recombinant LvAST exhibited significantly lower mortality and longer survival time in experimental infections by WSSV. In contrast, shrimps whose LvAST gene expression had been inhibited by RNA interference showed significantly higher WSSV infection intensity and shorter survival time following viral challenges. These results suggested that LvAST and WSSV both likely use ATPsyn-β as a receptor and LvAST plays a role in shrimp defense against WSSV infection. This represented the first research showing the involvement of astakines in host antiviral immunity. [ABSTRACT FROM AUTHOR]

Highlights: [•] White spot syndrome virus VP12 interacts with LvANT with dose-dependent manner. [•] LvANT was commonly found in all the tissues detected. [•] LvANT was colocalized with mitochondrion in shrimp hemocytes. [•] Injection of LvANT antiserum decreased the cumulative mortality of shrimp challenged by WSSV. [Copyright &y& Elsevier]

• The stain calcofluor white was used to detect Enterocytozoon hepatopenaei spores. • Spores were detected in spore suspensions, infected tissues, and feces. • Calcofluor white staining of feces is a noninvasive EHP monitoring method. A fluorescent stain, calcofluor white (CFW), was used for detection of spores of the microsporidium Enterocytozoon hepatopenaei (EHP). EHP spores in suspension, in feces, or in the infected hepatopancreas of the shrimp Penaeus vannamei , can be easily stained with this chitin and cellulose binding dye to show distinct blue-white fluorescent oval walls. The dye does not stain the host tissues. EHP spores showed orange-red spots by staining with hematoxylin and phloxine (H&P) in the section. CFW staining provides a simple and rapid method for determining the presence of EHP spores in fecal or tissue samples. [ABSTRACT FROM AUTHOR]