Simple immunoblot and immunohistochemical detection of Penaeus stylirostris densovirus using monoclonal antibodies to viral capsid protein expressed heterologously

Penaeus stylirostris densovirus (PstDNV), called formerly infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an important shrimp pathogen which can cause mortality in the blue shrimp Penaeus (Litopenaeus) stylirostris and stunting in the whiteleg shrimp Penaeus (Litopenaeus) vannamei. Five monoclonal antibodies (MAbs) were produced against the 37 kDa capsid protein 3 (CP3) of PstDNV expressed heterologously in the form of a fusion protein with glutathione-S-transferase called GST-CP3. All MAbs belonged to the IgG2b subclass and could bind to GST-CP3 at 300 pg/spot in immunodot-blot tests. They could detect CP3 in naturally infected shrimp extracts by Western blotting and dot blotting and in shrimp tissues by immunohistochemistry without cross-reactivity to extracts from uninfected shrimps or shrimps infected with several other viruses. Although dot blot assay sensitivity was approximately 1000 times lower than that of one step PCR for PstDNV, it easily detected PstDNV infections in field samples of Penaeus monodon and Penaeus vannamei.     

Role of vitelline envelope during fertilization in the black tiger shrimp, Penaeus monodon

Animal eggs possess investments through which sperm must penetrate. The aim of the present study was to investigate the role of the egg coating, the vitelline envelope, during sperm-egg interactions in the black tiger shrimp, Penaeus monodon. The site(s) of primary binding between sperm and egg and the possible binding molecule(s) for sperm were identified. In vitro adsorption of the vitelline envelope protein onto the sperm surface showed that primary binding occurred between the sperm anterior spike of acrosome intact sperm and the vitelline envelope. Results from streptavidin blotting revealed that the component of the vitelline envelope that interacts with the sperm integral membrane protein is a 370 kDa protein. In addition, it was shown that the vitelline envelope protein had no ability to induce acrosome reaction. These results suggest that the function of the vitelline envelope is as a primary binding site for sperm in shrimp, but not a sole trigger for the acrosome reaction.     

Molecular cloning and characterization of prophenoloxidase in the black tiger shrimp, Penaeus monodon.

A cDNA encoding shrimp, Penaeus monodon, prophenoloxidase (proPO) was obtained by screening a hemocyte library by plaque hybridization using a proPO cDNA fragment from freshwater crayfish, Pacifastaceus leniusculus, as a probe. The 3,002 bp cDNA contains an open reading frame of 2,121 bp and a 881 bp 3'-untranslated region. The molecular mass of the deduced amino acid sequence (688 amino acids) is 78,700 Da with an estimated pI of 5.8. Two putative copper binding sites are present and they have a highly conserved sequence around these sites. No signal peptide was detected in the shrimp proPO, as has been previously shown to be the case for all arthropod proPOs cloned so far. The cleavage site of zymogen activation is likely to be between Arg 44 and Val 45. A tentative complement-like motif (GCGWPQHM) is also present. Shrimp proPO mRNA is synthesized in the hemocytes and not in the hepatopancreas. Comparison of amino acid sequences showed that shrimp proPO is more closely related to another crustacean proPO, namely crayfish, than to the insect proPOs.     

Localization of the excitable membrane in the myelinated giant fiber of shrimp Penaeus japonicus

A significant DC-potential was recorded from the axon of shrimp myelinated giant nerve fiber, using a dye-filled microelectrode. Tracing of the dye assured that the source of the potential was the axonal membrane. All-or-none action potentials were obtained in the same preparation from which the ends of the axon were removed except for the synaptic region. It was concluded that the functionally excitable membrane is localized only in the synaptic region.     

Molecular cloning and characterization of an inhibitor of apoptosis protein (IAP) from the tiger shrimp, Penaeus monodon

The inhibitor of apoptosis proteins (IAPs) play important roles in both apoptosis and innate immunity. Here, we report the first cloning and characterization of a novel IAP family member, PmIAP, from Penaeus monodon. The full-length PmIAP cDNA is 4769bp, with an ORF encoding a protein of 698 amino acids. The PmIAP protein contains three BIR domains and a C-terminal RING domain, and its mRNA was expressed in all analyzed tissues. In insect cells, PmIAP, together with Spodoptera frugiperda IAP, AcMNPV P35, and WSSV449 (or ORF390, an anti-apoptosis protein encoded by white spot syndrome virus), could all block the apoptosis induced by Drosophila Reaper protein (Rpr), whereas only P35 and WSSV449 could block the apoptosis induced by actinomycin D. Co-immunoprecipitation showed that PmIAP physically interacted with Rpr, and in an immunofluorescent analysis the two proteins produced co-localized punctate signals in the cytoplasm. Deletion analysis revealed that both the BIR2 and BIR3 domains of PmIAP could independently bind to and inhibit Rpr, whereas the BIR1 domain could not. These results strongly suggest that PmIAP blocks Rpr's pro-apoptotic activity through mechanisms that are evolutionarily conserved across crustaceans, insects, and mammals.     

Binding of PmClipSP2 to microbial cell wall components and activation of the proPO-activating system in the black tiger shrimp Penaeus monodon

Clip domain serine proteinases (ClipSPs) play an important role in the prophenoloxidase-activating (proPO) system. In the shrimp Penaeus monodon, the ClipSP PmClipSP2 has been previously shown to bind to microbial polysaccharides (LPS and β-1,3-glucan) and likely activates the proPO system. To reveal the binding site of the PmClipSP2 protein, the N-terminal clip domain (Clip-PmClipSP) and C-terminal SP domain (SP-PmClipSP2) were separately cloned. The recombinant proteins were then assayed for their binding properties and involvement in proPO activation. According to the ELISA-based binding assay, rSP-PmClipSP2, but not rClip-PmClipSP, can bind immobilized LPS and β-1,3-glucan as well as significantly activate PO activity. The binding site at the SP domain is proposed to have a pattern sequence (X-[PFY]-X-[AFILV]-[AFY]-[AITV]-X-[ILV]-X(5)-W-[IL]-X) that is located at the C-terminal region of the SP domain of PmClipSP2. Deletion of the pattern sequence abolished binding to LPS and β-1,3-glucan. Conversely, a recombinant protein containing the pattern sequence (rPT-PmClipSP2-TRX) had the ability to bind to cell wall components, confirming that the pattern sequence at the C-terminus of PmClipSP2 is responsible for binding to microbes, subsequently leading to activation of the proPO cascade.     

Localization of anti-lipopolysaccharide factor (ALFPm3) in tissues of the black tiger shrimp, Penaeus monodon, and characterization of its binding properties

Anti-lipopolysaccharide factor (ALF) is an antimicrobial peptide originally identified from horseshoe crabs and recently found in several shrimp species. ALFPm3, the most abundant isoform in the black tiger shrimp, Penaeus monodon, has been shown to possess a broad spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria, and filamentous fungi. In this study, a potential role for ALFPm3 in the shrimp innate immunity was revealed by examining the distribution of the protein in shrimp tissues in response to Vibrio harveyi challenge. Immunohistochemistry using anti-ALFPm3 antibody showed that the ALFPm3 protein is primarily localized in hemocytes and the positive cells observed at the injection site and in the cephalothorax are infiltrating hemocytes that migrate into shrimp tissues after bacterial injection. A rapid increase in the number of hemocytes producing ALFPm3 observed in V. harveyi-injected shrimp suggests a likely important function of the protein in defense against invading pathogens. ALFPm3 was shown to be able to bind to Gram-negative and Gram-positive bacterial cells and their major cell wall components, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), respectively. The results suggested that ALFPm3 performs its antibacterial activity by binding to component(s) of the bacterial cell wall.     

Vitreous fluid sampling and viral genome detection for the diagnosis of viral retinitis in patients with AIDS

Cytomegalovirus (CMV) causes severe necrotizing retinitis in patients with the acquired immune deficiency syndrome (AIDS) and other herpesviruses have been implicated in the acute retinal necrosis syndrome (ARN), seen in both the immunocompetent and the immunosuppressed. At present the diagnosis of viral retinitis relies solely on clinical appearances. In order to assess whether the detection of herpesvirus-specific DNA in cell-free vitreous biopsy samples could be useful in the early diagnosis of viral retinitis, vitreous fluid samples were taken from 100 patients. Fifty patients had AIDS as defined by the Centers for Disease Control, (MMWR 36 (suppl 1S):1S–15S, 1987) and retinal disease. The remainder were not known to be HIV infected and had no clinical evidence of retinal infection. Each sample was tested for the presence of CMV, herpes simplex virus 1 (HSV-1), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV6), by amplification of viral DNA using a sensitive and specific nested poly-merase chain reaction (PCR). The presence of detectable CMV or VZV DNA was clearly associated with clinical disease whereas the presence of HSV-1, EBV, and HHV6 sequences were not. Clinical discrimination between CMV- and VZV-associated retinitis was greatly enhanced when the PCR results were taken into consideration. © 1994 Wiley-Liss, Inc.     

Expression and characterisation of tiger shrimp Penaeus monodon penaeidin (mo-penaeidin) in various tissues, during early embryonic development and moulting stages

A penaeidin family, mo-penaeidin was cloned from the haemocytes of tiger shrimp Penaeus monodon using genomic polymerase chain reaction (PCR) by gene specific primers. Analysis of nucleotide sequence revealed that this mo-penaeidin consists of 1348 bp containing one intron (680 bp) and two exons (210 and 458 bp). It has an open reading frame (ORF) of 222 p, which encodes a protein of 74 amino acids including a signal peptide of 19 amino acids. The calculated molecular mass of the mature protein (55 amino acids) is 6.059 kDa with an estimated pI of 9.3. The deduced amino acid sequence of mo-penaeidin has similarity to that of penaeidin from Fenneropenaeus chinensis (73%), Farfantepenaeus paulensis (66%), Litopenaeus schmitti (53-67%), L. stylirostris (50-67%), L. setiferus (50-62%), L. vannamei (44-66%), and Marsupenaeus japonicus (33%), respectively. Phylogenetic tree analysis indicated that penaeidin (including mo-penaeidin, penaeidin, and penaeidin 5, 2, 3k, 3c1) of P. monodon is distinct from penaeidin 1, penaeidin 2, penaeidin 3 and penaeidin 4 of other penaeid shrimps. The mo-penaeidin mRNA was detected in various tissues including ovary and mandibular organ. The mo-penaeidin mRNA was present in one cell to postlarva stage with higher level at nauplius I (9h post hatching) and higher expression during the intermoult stage indicating an early innate immunity and different immunity at moulting stage.     

Human herpesvirus 6 DNA levels in cerebrospinal fluid due to primary infection differ from those due to chromosomal viral integration and have implications for diagnosis of encephalitis

The prevalence and concentration of human herpesvirus 6 (HHV-6) DNA in the cerebrospinal fluid (CSF) of the immunocompetent in primary infection was compared with that in viral chromosomal integration. Samples from 510 individuals with suspected encephalitis, 200 young children and 310 older children and/or adults, and 12 other patients were tested. HHV-6 DNA concentration (log(10) copies/ml) was measured in CSF, serum, and whole blood using PCR. Serum HHV-6 immunoglobulin G antibody was measured by indirect immunofluorescence. Primary infection was defined by antibody seroconversion and/or a low concentration of HHV-6 DNA (<3.0 log(10) copies/ml) in a seronegative serum. Chromosomal integration was defined by a high concentration of viral DNA in serum (>/=3.5 log(10) copies/ml) or whole blood (>/=6.0 log(10) copies/ml). The prevalences of CSF HHV-6 DNA in primary infection and chromosomal integration were 2.5% and 2.0%, respectively, in the young children (<2 years) and 0% and 1.3%, respectively, in the older children and/or adults. The mean concentration of CSF HHV-6 DNA in 9 children with primary infection (2.4 log(10) copies/ml) was significantly lower than that of 21 patients with viral chromosomal integration (4.0 log(10) copies/ml). Only HHV-6B DNA was found in primary infection, whereas in viral integration, 4 patients had HHV-6A and 17 patients HHV-6B. Apart from primary infection, chromosomal integration is the most likely cause of HHV-6 DNA in the CSF of the immunocompetent. Our results show that any diagnosis of HHV-6 encephalitis or other type of active central nervous system infection should not be made without first excluding chromosomal HHV-6 integration by measuring DNA load in CSF, serum, and/or whole blood.