Interior modification of Macrobrachium rosenbergii nodavirus-like particle enhances encapsulation of VP37-dsRNA against shrimp white spot syndrome infection.

BACKGROUND: Application of a virus-like particle (VLP) as a nanocontainer to encapsulate double stranded (ds)RNA to control viral infection in shrimp aquaculture has been extensively reported. In this study, we aimed at improving VLP's encapsulation efficiency which should lead to a superior fighting weapon with disastrous viruses. RESULTS: We constructed 2 variants of chimeric Macrobrachium rosenbergii nodavirus (MrNV)-like particles (V1- and V2-MrN-VLPs) and tested their efficiency to encapsulate VP37 double stranded RNA as well as WSSV protection in P. vannamei. Two types of short peptides, RNA-binding domain (RBD) and deca-arginine (10R) were successfully engineered into the interior surface of VLP, the site where the contact with VP37-dsRNA occurs. TEM and dynamic light scattering (DLS) analyses revealed that the chimeric VLPs remained their assembling property to be an icosahedral symmetric particle with a diameter of about 30 nm, similar to the original MrN-VLP particle. The superior encapsulation efficiency of VP37-dsRNA into V2-MrN-VLP was achieved, which was slightly better than that of V1-MrN-VLP but far better (1.4-fold) than its parental V0-MrN-VLP which the mole ratio of 7.5-10.5 for all VLP variants. The protection effect against challenging WSSV (as gauged from the level of VP37 gene and the remaining viral copy number in shrimp) was significantly improved in both V1- and V2-MrN-VLP compared with an original V0-MrN-VLP template. CONCLUSION: MrN-VLP (V0-) were re-engineered interiorly with RBD (V1-) and 10R (V2-) peptides which had an improved VP37-dsRNA encapsulation capability. The protection effect against WSSV infection through shrimp administration with dsRNA + V1-/V2-MrN VLPs was experimentally evident.

Confirmatory test of active IHHNV infection in shrimp by immunohistochemistry and IHHNV-LongAmp PCR.

The presence of endogenous viral elements (EVE) in the penaeid shrimp genome has been recently reported and suggested to be involved in the host recognition of viral invaders. Our previous report of a search for EVE of infectious hypodermal and haematopoietic necrosis virus (IHHNV-EVE) in the Thai Penaeus monodon whole genome sequence project (GenBank accession no. JABERT000000000) confirmed the presence of three clusters of EVE derived from IHHNV in the shrimp genome. This study aimed to compare an immunohistochemistry method (IHC) and a PCR method to detect infectious IHHNV infection in shrimp. First, specimens collected from farms were checked for IHHNV using three PCR methods; two methods were recommended by WOAH (309 and 389 methods), and a newly established long-range PCR for IHHNV (IHHNV-LA PCR) targeting almost the whole genome (>90%) of IHHNV. Among 29 specimens tested, 24 specimens were positive for WOAH methods (at least one method). Among 24 WOAH-positive specimens (WOAH+), there were 18 specimens with positive IHHNV-LA PCR method (WOAH+/LA+), six specimens with negative IHHNV-LA PCR method (WOAH+/LA-). Six specimens were negative for all methods (WOAH-/LA-). The positive signals detected by IHC method were found only in the specimens with WOAH+/LA+. The results suggest that the WOAH+/LA- specimens were not infected with IHHNV, and the positive WOAH method might result from the EVE-IHHNV. The study recommends combining the IHHNV-LA PCR method and IHC with positive PCR results from WOAH's recommended methods to confirm IHHNV infection.

Sesquiterpenoid pathway in the mandibular organ of Penaeus monodon: Cloning, expression, characterization of PmJHAMT and its alteration response to eyestalk ablation.

Methyl farnesoate (MF), a crustacean equivalent of juvenile hormone (JH) of insects, is known to be produced from the mandibular organ (MO). This study reports transcriptome analysis of Penaeus monodon MO and identifies putative genes encoding enzymes in the sesquiterpenoid pathway. A total of 44,490,420 clean reads were obtained and utilized for subsequent analysis. De novo assembly created 31,201 transcripts and 31,167 unigenes. To archive the functional annotation, all unigenes were annotated with KOG, KEGG, and GO. Putative genes encoding enzymes and regulatory proteins involved in the sesquiterpenoid pathway were obtained from the MO transcriptome data based on the conserved domains and sequence homology. They included S-adenosylmethionine synthetase, farnesyl pyrophosphate synthase, short chain dependent dehydrogenase/reductase (SDR), NAD(P) + -dependent aldehyde dehydrogenase, S-adenosylmethionine-dependent methyltransferases or juvenile hormone acid-O-methyl transferase (JHAMT), farnesoic acid O-methyl transferase (FAMeT), juvenile hormone binding protein, cytochrome C/P-450 family 15 (CRYP15A1)/methylfarnesoate epoxidase (MFE), juvenile hormone epoxide hydrolase (JHEH), and juvenile hormone esterase (JHE). We first identified and characterized JHAMT orthologs inP. monodon(PmJHAMT). The complete cDNA sequence ofPmJHAMTconsisted of 1,221 nt encoded 271 amino acids with a conserved S-adenosyl methionine (SAM) binding domain. Phylogenetic analysis clusteredPmJHAMTinto the group JHAMT with the same clade of the crabPortunus trituberculausJHAMT. Moreover, the predicted three-dimensional structure of PmJHAMT showed remarkable similarity with the recent crystal structure ofthe Bombyx moriJHAMT homodimer. RT-PCR analysis revealed that PmJHAMT was exclusively expressed in MO and initially expressed at stage 3 postlarvae. In situ hybridization with a specific probe to PmJHAMT validated the specific expression of this gene in MO cells. Finally, we evaluated the regulation of MO by eyestalk inhibitory peptides. Diminishing MO inhibitory hormone through unilateral eyestalk ablation resulted in a significantly higher expression ofPmJHAMTin MO by quantitative PCR. This result indicated that the eyestalk inhibitory hormone inhibited MF synthesis byPmJHAMTgene suppression in the MO. This finding provides insight into the crustacean sesquiterpenoid pathway and improves our understanding of crustacean endocrinology.

Viral Capsid Change upon Encapsulation of Double-Stranded DNA into an Infectious Hypodermal and Hematopoietic Necrosis Virus-like Particle.

In this study, we aimed to encapsulate the sizable double-stranded DNA (dsDNA, 3.9 kbp) into a small-sized infectious hypodermal and hematopoietic necrosis virus-like particle (IHHNV-VLP; T = 1) and compared the changes in capsid structure between dsDNA-filled VLP and empty VLP. Based on our encapsulation protocol, IHHNV-VLP was able to load dsDNA at an efficiency of 30-40% (w/w) into its cavity. Structural analysis revealed two subclasses of IHHNV-VLP, so-called empty and dsDNA-filled VLPs. The three-dimensional (3D) structure of the empty VLP produced in E. coli was similar to that of the empty IHHNV-VLP produced in Sf9 insect cells. The size of the dsDNA-filled VLP was slightly bigger (50 Å) than its empty VLP counterpart; however, the capsid structure was drastically altered. The capsid was about 1.5-fold thicker due to the thickening of the capsid interior, presumably from DNA-capsid interaction evident from capsid protrusions or nodules on the interior surface. In addition, the morphological changes of the capsid exterior were particularly observed in the vicinity of the five-fold axes, where the counter-clockwise twisting of the "tripod" structure at the vertex of the five-fold channel was evident, resulting in a widening of the channel's opening. Whether these capsid changes are similar to virion capsid maturation in the host cells remains to be investigated. Nevertheless, the ability of IHHNV-VLP to encapsulate the sizable dsDNA has opened up the opportunity to package a dsDNA vector that can insert exogenous genes and target susceptible shrimp cells in order to halt viral infection.

Infectious hypodermal and hematopoietic necrosis virus-like particle (IHHNV-VLP) induces peroxiredoxin expression and activity in Fenneropenaeus merguiensis.

Virus like particles (VLPs) are non-infectious nanoparticles containing repetitive, high density viral epitopes on the surface and can prevent viral infections in aquatic animals. Here, we evaluated the immuno-stimulation effect of infectious hypodermal and hematopoietic necrosis virus like particle (IHHNV-VLP) using a next generation sequencing in Fenneropenaeus merguiensis to identify the important immune-related genes that may prevent viral infection. The in situ target of IHHNV was predominantly found in gill tissue following IHHNV-VLP administration in juvenile shrimp. Comparative transcriptome analysis in the injected gills showed that there were 326 unigenes expressed differently than the mock-injected samples. One of the most differential genes between the two animal groups was the antioxidative gene, peroxiredoxin (FmPrx), that was up-regulated after 6 h post-VLP injection. Phylogenetic tree analysis showed that this gene could be found among many shrimp species and was closely clustered among Prx families. The expression of FmPrx was also detected in all tissues examined, thus suggesting the multi-functional roles of this gene in many tissues. Administration of IHHNV-VLP in vivo led to a significant increase in peroxidase activity in gill tissue-approximately two-fold versus control animals; the WSSV copy number was significantly reduced. These data suggest that IHHNV-VLP exerts an immune-stimulating effect by enhancing the level of immune-related genes including FmPrx and its corresponding peroxidase activity, which are a well-known part of the shrimp innate immune system.

Suppression of a Novel Vitellogenesis-Inhibiting Hormone Significantly Increases Ovarian Vitellogenesis in the Black Tiger Shrimp, Penaeus monodon.

In this study, a novel Crustacean Hyperglycemic Hormone-type II gene (CHH-type II) was identified and biologically characterized in a shrimp, Penaeus monodon. Based on its structure and function, this gene was named P. monodon vitellogenesis-inhibiting hormone (PemVIH). The complete cDNA sequence of PemVIH consisted of 1,022 nt with an open reading frame (ORF) of 339 nt encoding a polypeptide of 112 amino acids. It was classified as a member of the CHH-type II family based on conserved cysteine residues, a characteristically positioned glycine residue, and the absence of CHH precursor-related peptide (CPRP) domain. The deduced mature PemVIH shared the highest sequence similarities with giant river prawn sinus gland peptide A. Unlike P. monodon gonad-inhibiting hormone (PemGIH), PemVIH was expressed only in the brain and ventral nerve cord, but not the eyestalks. Whole mount immunofluorescence using a newly generated PemVIH antiserum detected positive signals in neuronal cluster 9/11 and 17 of the brain, commissural ganglion (CoG), and neuronal clusters of ventral nerve cord. The presence of PemVIH-positive neurons in CoG, a part of stomatogastric nervous system, suggested a potential mechanism for crosstalk between nutritional and reproductive signaling. The role of PemVIH in vitellogenesis was evaluated using RNA interference technique. Temporal knockdown of PemVIH in female subadults resulted in a 3-fold increase in ovarian vitellogenin expression, suggesting an inhibitory role of PemVIH in vitellogenesis. This study provided novel insight into the control of vitellogenesis and additional strategies for improving ovarian maturation in P. monodon without the current harmful practice of eyestalk ablation.

Chimeric virus-like particles (VLPs) designed from shrimp nodavirus (MrNV) capsid protein specifically target EGFR-positive human colorectal cancer cells.

Recombinant MrNV capsid protein has been shown to effectively deliver plasmid DNA and dsRNA into Sf9 insect cells and shrimp tissues. To extend its application to cancer cell-targeting drug delivery, we created three different types of chimeric MrNV virus-like particles (VLPs) (R-MrNV, I-MrNV, and E-MrNV) that have specificity toward the epidermal growth factor receptor (EGFR), a cancer cell biomarker, by incorporating the EGFR-specific GE11 peptide at 3 different locations within the host cell recognition site of the capsid. All three chimeric MrNV-VLPs preserved the ability to form a mulberry-like VLP structure and to encapsulate EGFP DNA plasmid with an efficiency comparable to that previously reported for normal MrNV (N-MrNV). Compared to N-MrNV, the chimeric R-MrNV and E-MrNV carrying the exposed GE-11 peptide showed a significantly enhanced binding and internalization abilities that were specific towards EGFR expression in colorectal cancer cells (SW480). Specific targeting of chimeric MrNV to EGFR was proven by both EGFR silencing with siRNA vector and a competition with excess GE-11 peptide as well as the use of EGFR-negative colorectal cells (SW620) and breast cancer cells (MCF7). We demonstrated here that both chimeric R-MrNV and E-MrNV could be used to encapsulate cargo such as exogenous DNA and deliver it specifically to EGFR-positive cells. Our study presents the potential use of surface-modified VLPs of shrimp virus origin as nanocontainers for targeted cancer drug delivery.

Establishment of hematopoietic tissue primary cell cultures from the giant freshwater prawn Macrobrachium rosenbergii.

The giant freshwater prawn Macrobrachium rosenbergii is one of the most important aquaculture species in Southeast Asia. In this study, in vitro culture of its hematopoietic tissue cells was achieved and characterized for use as a tool to study its pathogens that cause major farm losses. By transmission electron microscopy, the ultrastructure of the primary culture cells was similar to that of cells lining intact hematopoietic tissue lobes. Proliferating cell nuclear antigen (PCNA) (a marker for hematopoietic stem cell proliferation) was detected in some of the cultured cells by polymerase chain reaction (PCR) testing and flow cytometry. Using a specific staining method to detect phenoloxidase activity and using PCR to detect expression markers for semigranular and granular hemocytes (e.g., prophenoloxidase activating enzyme and prophenoloxidase) revealed that some of the primary cells were able to differentiate into mature hemocytes within 24 h. These results showed that some cells in the cultures were hematopoietic stem cells that could be used to study other interesting research topics (e.g. host pathogen interactions and development of an immortal hematopoietic stem cell line).

Dual VP28 and VP37 dsRNA encapsulation in IHHNV virus-like particles enhances shrimp protection against white spot syndrome virus.

Accumulative evidence of using double stranded (ds) RNA encapsulated into virus like particle (VLP) nanocarrier has open feasibility to fight against shrimp viral infection in aquaculture field. In this study, we co-encapsulated VP37 and VP28 dsRNA into hypodermal and hematopoietic necrosis virus (IHHNV) like particle and investigated its protection against white spot syndrome virus (WSSV). Five micrograms of each dsRNA were used as starting materials to load into VLP, while the loading efficiency was slightly different, i.e, VP37 dsRNA had somewhat a better load into VLP's cavity. It was apparent that co-encapsulation of dual dsRNA showed a superior WSSV silencing ability than the single dsRNA counterpart as evidence by the lower WSSV gene expression and its copy number in the gill tissues. Besides, we also demonstrated that co-encapsulated dual dsRNA into IHHNV-VLP stimulated the increased number of hemocytes and the corresponding PO activity as well as up-regulated proPO gene expression in hemocytes to resist viral invasion after an acute stage of WSSV infection. This synergistic action of dual dsRNA encapsulated into IHHNV-VLPs could thus act to delay time of shrimp death and reduced shrimp cumulative mortality greater than the single, naked dsRNA treatment and positive control groups. The obtaining results would encourage the feasibility to use it as a new weapon to fight WSSV infection in shrimp aquaculture.

Immunopathogenesis of hematopoietic tissues in response to Vibrio parahaemolyticus (VPAHPND) infection in Macrobrachium rosenbergii.

In crustacean, hemocytes are known as crucial components of crustaceans' innate immunity against pathogens. Drastic hemocytes reduction during infectious disease is apparently related to disease severity and calls for a health status evaluation and aquaculture management. The molecular pathogenesis of hemocytes loss during bacterial infection was elucidated with VPAHPND challenged in M. rosenbergii. We report herein a correlation between hemocyte loss and the pathogenicity and aggressive immune response in hematopoietic tissues of moribund M. rosenbergii. In this study, adult freshwater prawn was administered an LC50 dose of VPAHPND; bacterial clearance ensued, and success was reached within 24 h. Hemocytes increased in survival, yet drastically decreased in moribund prawn. Pathological analysis of hematopoietic tissue of moribund prawn showed apparent abnormal signs, including the presence of bacteria, a small number of mitotic cells, cellular swelling, loosening of connective tissue, and karyorrhectic nuclei cells. A significant upregulation of a core apoptotic machinery gene, caspase-3, was detected in hematopoietic tissue of moribund shrimp, but not in those of Escherichia coli DH5α (non-pathogenic bacteria) and VPAHPND survival prawn. The highest level was found in the moribund group, which confirms the occurrence of apoptosis in this hematopoietic tissue. Further, our results suggest that hematopoietic tissue damage may arise from inflammation triggered by an aggressive immune response. Immune activation was indicated by the comparison of immune-related gene expression between controls, E. coli (DH5α)-infected (non-pathogenic), and VPAHPND-infected survival groups with moribund prawn. RT-PCR revealed a significant upregulation of all genes in hematopoietic tissues and hemocytes within 6-12 h and declined by 24 h. This evident related to the almost VPAHPND are clearance in survival and E. coli (DH5α) challenged group in contrast with drastic high expression was determined in moribund group. We conclude that a reduction of renewing circulating hemocytes in fatally VPAHPND-infected prawn was caused by an acute self-destructive immune response by hematopoietic cells.

The hematopoietic organ of Macrobrachium rosenbergii: Structure, organization and immune status.

The hematopoietic organ (HO) of the giant freshwater prawn Macrobrachium rosenbergii is a discrete, whitish mass located in the epigastric region of the cephalothorax, posterior to the brain. It is composed of hematopoietic cells arranged in a thick layer of numerous lobules that surround a central hemal sinus from which they are separated by a thin sheath. At the center of the sinus is the muscular cor frontale. The lobules extend radially outward from the sinus in three developmental zones. Basal Zone 1 nearest the sinus contains large hematopoietic stem cells with euchromatic nuclei that stain positive for proliferation cell nuclear antigen (PCNA). Zone 2 contains smaller, actively dividing cells as indicated by positive 5-bromo-20-deoxyuridine (BrdU) staining. Distal Zone 3 contains small, loosely packed cells with heterochromatic nuclei, many cytoplasmic granules and vesicles indicating that they will eventually differentiate into hemocytes and enter circulation. Three main arteries, namely the ophthalmic and the 2 branches of the antennary, connect the heart to the HO. Use of India ink and 0.1 µm fluorescent micro-beads injected into the heart revealed that the cor frontale could immediately remove foreign particles from hemolymph by filtration. Fluorescent beads were also detected in the hematopoietic tissue at 30 min after injection, indicating that it could be penetrated by foreign particles. However, the fluorescent signal completely disappeared from the whole HO after 4 h, indicating its role in removal of foreign particles. In conclusion, the present study demonstrated for the first time the detailed histological structures of the HO of M. rosenbergii and its relationship to hematopoiesis and removal of foreign particles from hemolymph.

Existence of an egg-laying hormone-like peptide in male reproductive system of the giant freshwater prawn, Macrobrachium rosenbergii.

The giant freshwater prawn, Macrobrachium rosenbergii, is an important aquaculture species. A better understanding of the molecular components of reproduction in this species would help to advance the prawn production. In the present study, we demonstrated the presence of an egg laying hormone (ELH)-like peptide in the male reproductive system. First, an antibody to the abalone (a)ELH was generated, and by Western blot it was shown to specifically bound to a protein from the male M. rosenbergii reproductive tissues with a similar size to molluscan ELH. This aELH-like peptide was localized in spermatogonia in the testes of all three male morphotypes: blue claw, orange claw and small males. Moreover, the aELH-like peptide was detected in the epithelium of the spermatic duct and its associated smooth muscle cell layers and on the outer surface of spermatozoa. As well, the aELH-like peptide was detected in the spermatophore located in the female thelycum at 4-6 h post-mating, indicating that it was transferred to the female during copulation. Taken together, we suggest that this aELH-like peptide could be as a male inducing factor that helped to accelerate female spawning. Liquid chromatography of crude extracts and immunoblot analysis suggested that the aELH-like peptide could be further purified for ultimate characterization.

Hematopoietic tissue of Macrobrachium rosenbergii plays dual roles as a source of hemocyte hematopoiesis and as a defensive mechanism against Macrobrachium rosenbergii nodavirus infection.

White tail disease caused by Macrobrachium rosenbergii nodavirus (MrNV) infection takes place only in nauplii, not adults, of M. rosenbergii prawn. Hemocyte homeostasis and immune-related functions derived from the hematopoietic tissue (Hpt) in adult prawn are presumed to play roles in resisting viral infection. To elucidate the role of the Hpt cell response to MrNV, a comparative transcriptome analysis was performed with MrNV-infected prawn at various time intervals. The results showed that there were 462 unigenes that were differentially expressed between mock and infected samples. BlastX sequence analysis revealed that two proteins, crustacean hematopoietic factor (CHF) and cell growth-regulating zinc finger protein (Lyar), are involved in hemocyte hematopoiesis and are up-regulated during MrNV infection. In fact, genes involved in cell growth regulation and immunity were highly expressed at 6 h and decreased within 24 h post-infection. Localization studies in the Hpt tissue revealed the presence of anti-lipopolysaccharide factor (ALF) and CHF mRNAs in Hpt cells. Considering these findings, we concluded that resistance to MrNV infection in adult prawn is due to an increase in humoral immune factors and the acceleration of hemocyte homeostasis by the dual roles of the Hpt organ in M. rosenbergii.

Nanocontainer designed from an infectious hypodermal and hematopoietic necrosis virus (IHHNV) has excellent physical stability and ability to deliver shrimp tissues.

BACKGROUND: A virus-like particle (VLP) is an excellent tool for a compound delivery system due to its simple composition, symmetrical structure and self-assembly. Its surface modification both chemically and genetically is established, leading to the target-specific delivery and improved encapsulation efficiency. However, its physical stabilities against many harsh conditions that guarantee long term storage and oral administration have been much less studied. METHODS: IHHNV-VLPs were reconstructed from recombinant IHHNV capsid protein in E. coli. Their physical properties against three strong physical conditions including long term storage (0-30 days) in 4 °C, physical stabilities against broad ranged pH (4-9) and against three types of digestive enzymes were tested. Disassembly and reassembly of VLPs for encapsidating an enhanced green fluorescent protein tagged plasmid DNA (EGFP-VLPs) were controlled by the use of reducing agent (DTT) and calcium specific chelating agent (EGTA). Lastly, delivering ability of EGFP-VLPs was performed in vivo by intramuscular injection and traced the expression of GFP in the shrimp tissues 24 hr post-injection. RESULTS: Upon its purification, IHHNV-VLPs were able to be kept at 4 °C up to 30 days with only slight degradation. They were very stable in basic condition (pH 8-9) and to a lesser extent in acidic condition (pH 4-6) while they could stand digestions of trypsin and chymotrypsin better than pepsin. As similar with many other non-enveloped viruses, the assembly of IHHNV-VLPs was dependent on both disulfide bridging and calcium ions which allowed us to control disassembly and reassembly of these VLPs to pack EGFP plasmid DNA. IHHNV-VLPs could deliver EGFP plasmids into shrimp muscles and gills as evident by RT-PCR and confocal microscopy demonstrating the expression of GFP in the targeted tissues. DISCUSSION: There are extensive data in which capsid proteins of the non-enveloped viruses in the form of VLPs are constructed and used as nano-containers for therapeutic compound delivery. However, the bottleneck of its application as an excellent delivery container for oral administration would rely solely on physical stability and interacting ability of VLPs to the host cells. These properties are retained for IHHNV-VLPs reported herein. Thus, IHHNV-VLPs would stand as a good applicable nanocontainer to carry therapeutic agents towards the targeting tissues against ionic and digestive conditions via oral administration in aquaculture field.

Scabraside D Extracted from Holothuria scabra Induces Apoptosis and Inhibits Growth of Human Cholangiocarcinoma Xenografts in Mice.

Scabraside D, a sulfated triterpene glycoside extract from sea cucumber Holothulia scabra, shows various biological activities, but effects on human cholangiocarcinoma cells have not previously been reported. In the present study, we investigated the activity of scabraside D against human cholangiocarcinoma (HuCCA) both in vitro and for tumor growth inhibition in vivo using a xenograft model in nude mice. Scabraside D (12.5-100 µg/mL) significantly decreased the viability and the migration of the HuCCA cells in a dose-dependent manner, with 50% inhibitory concentration (IC50) of 12.8 ± 0.05 µg/mL at 24 h. It induced signs of apoptotic cells, including shrinkage, pyknosis and karyorrhetic nuclei and DNA fragmentation on agarose gel electrophoresis. Moreover, by quantitative real-time PCR, scabraside D effectively decreased Bcl-2 while increasing Bax and Caspase-3 gene expression levels suggesting that the scabraside D could induce apoptosis in HuCCA cells. In vivo study demonstrated that scabraside D (1 mg/kg/day, i.p. for 21 days) significantly reduced growth of the HuCCA xenografts without adverse effects on the nude mice. Conclusively, scabraside D induced apoptosis in HuCCA cells and reduced the growth of HuCCA xenographs model. Therefore, scabraside D may have potential as a new therapeutic agent for cholangiocarcinoma.

Production and characterization of a monoclonal antibody against recombinant cathepsin L1 of Fasciola gigantica.

Monoclonal antibodies (MoAbs) against a recombinant cathepsin L1 of Fasciola gigantica (rFgCatL1) were produced in vitro by fusion of BALB/c mice spleen cells immunized with rFgCatL1 and mouse myeloma cells. Reactivity and specificity of these MoAbs were evaluated by indirect ELISA and immunoblotting techniques. Seven MoAb clones were selected from the stable hybridoma clones, namely 1E10, 1F5, 3D11, 4B10, 4D3, 4E3 and 5E7. Clones 1E10, 1F5 and 3D11 were IgM, whereas clones 4B10, 4D3, 4E3 and 5E7 were IgG1. All MoAbs had kappa light chain isotypes. All MoAbs reacted with rCatL1 at molecular weight (MW) 30kDa and with the native CatL1 at MW 27kDa in whole body (WB) extracts of metacercariae (Met), newly excysted juveniles (NEJ), 1, 3, 5-week-old juveniles (Ju), adult WB and adult excretory-secretory (ES) fractions, but not with adult tegumental antigens (TA). All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants and human, including Paramphistomum cervi, Eurytrema pancreaticum, Gigantocotyle explanatum, Schistosoma spindale, Schistosoma mansoni, Moniezia benedeni, Avitellina centripunctata, Trichuris sp., Haemonchus placei and Setaria labiato-papillosa. Localization of CatL1 in each developmental stages of F. gigantica by immunoperoxidase technique, using these MoAbs as probes, indicated that CatL1 was present at high concentration in the caecal epithelium and caecal lumen of metacercariae, NEJ, 1, 3, 5-week-old juveniles and adult fluke. This finding indicated that CatL1 is a copiously expressed parasite protein that is released into the ES, thus CatL1 and its MoAb could be a good candidate for immunodiagnosis of fasciolosis in ruminant and human.

Cathepsin D in human reproductive tissues: cellular localization in testis and epididymis and surface distribution in different sperm conditions.

Mammalian sperm surface antigens are acquired either during spermatogenesis or sperm maturation in the epididymis. These antigens, many of which are hydrolytic enzymes, are actively synthesized and secreted by the resident epithelial cells and adsorbed to the sperm membrane as part of posttesticular sperm modification. In this study, we aimed to investigate the expression of cathepsin-D (CAT-D) in human reproductive tissues and its distribution on the sperm surface in different sperm conditions. Immunohistochemical results revealed the expression of CAT-D in the somatic Sertoli and Leydig cells without showing any immunoreactivity in any germ cells, despite their engagement of the acrosomal system. A strong immunoreactivity of anti-CAT-D was also detected in the epididymal epithelium, chiefly in the principal cells, which are known to actively synthesize and secrete proteins into the epididymal lumen. The absence of CAT-D in the clear cells was unexpected because these cells are known to engage the endosomal machinery. We further showed that CAT-D was anchored on the sperm surface confined to the postacrosomal region without any lateral redistribution within the membrane during sperm capacitation. However, the enzyme underwent changes to be an active form of a 29/30-kd doublet during sperm capacitation. Using CAT-D as a marker, we were able to demonstrate here localization of the enzyme in human reproductive tissues, as well as reveal membrane modification in human sperm during maturation and capacitation processes.

Existence and distribution of gonadotropin-releasing hormone-like peptides in the central nervous system and ovary of the Pacific white shrimp, Litopenaeus vannamei.

We used antibodies against octopus gonadotropin-releasing hormone (octGnRH) and tunicate GnRH (tGnRH-I) in order to investigate the existence and distribution of GnRH-like peptides in the central nervous system (CNS) and in the ovary during various stages of the ovarian cycle of the white shrimp, Litopenaeus vannamei. OctGnRH-immunoreactive and tGnRH-I-immunoreactive neurons and fibers were present in several regions of the supraesophageal ganglion (brain), subesophageal ganglion (SEG), thoracic ganglia, and abdominal ganglia. In the brain, both octGnRH immunoreactivity (ir) and tGnRH-I-ir were detected in neurons of clusters 6, 11, 17, and associated fibers, and the anterior medial protocerebral, posterior medial protocerebral, olfactory, and tegumentary neuropils. In the SEG and thoracic ganglia, octGnRH-immunoreactive and tGnRH-I-immunoreactive neurons and fibers were present in dorsolateral and ventromedial cell clusters and in surrounding fibers. Only immunoreactive fibers were detected in the abdominal ganglia. In the ovary, both octGnRH and tGnRH-I were detected at medium intensity in the cytoplasm of early step oocytes (Oc2) and, at high intensity, in Oc3. Furthermore, octGnRH-ir and tGnRH-I-ir were intense in follicular cells surrounding Oc2 and Oc3. The presence of GnRH-ir in the CNS and ovary indicates that GnRH-like peptides occur in the white shrimp, and that GnRHs are involved in the reproductive process, especially ovarian maturation and the differentiation of oocytes, as reported in other species.

A virulent isolate of yellow head nidovirus contains a deformed envelope glycoprotein gp116.

Yellow head virus (YHV) is a highly virulent pathogen of penaeid shrimp. An isolate obtained from Penaeus vannamei during a yellow head disease outbreak in February 2006 in Ratchaburi Province, Thailand was purified following passage in experimentally infected shrimp. SDS-PAGE of purified virions indicated that envelope glycoprotein gp116 in the Ratchaburi/2006 isolate was smaller and relatively less abundant than in the Chachoengsao/1998 YHV reference strain. The variant gp116 reacted poorly in immunoblots with a gp116 mouse monoclonal antibody and a rabbit anti-serum to a baculovirus-expressed, C-terminally truncated, [His](6)-tagged gp116 that reacted strongly with gp116 of the homologous Chachoengsao/1998 strain. The anti-gp116 polyclonal serum also failed to neutralise the infectivity of the Ratchaburi/2006 isolate in in-vivo assays conducted in P. vannamei, but effectively neutralised the infectivity of the reference strain. Sequence analysis of the approximately 6.0 kb structural protein gene region and 3'UTR of the Ratchaburi/2006 isolate indicated >99.9% overall nucleotide identity with the Chachoengsao/1998 strain. However, in Ratchaburi/2006 a deletion in ORF3, corresponding to 54 amino acids near the N-terminal signal peptidase cleavage site of gp116, resulted in the loss of six conserved cysteine residues and two predicted N-glycosylation sites. Analysis of this ORF3 region in 25 viruses representing each of the six genotypes in the yellow head complex identified this modified form of gp116 in two other virulent YHV isolates classified as genotype 1b. The data indicate that, although the deletion causes a significant structural deformation of gp116 which reduces its incorporation into virions and eliminates the major neutralisation sites, the virus remains highly infectious, virulent and fit for survival.