Piper DNA virus 1 and 2 are endogenous pararetroviruses integrated into chromosomes of black pepper (Piper nigrum L).

A previous study named 7178 and 892 bp contigs obtained through high-throughput sequencing (HTS) of black pepper as Piper DNA virus 1 (PDV-1) and PDV-2 respectively. In the present study, HTS results were confirmed through polymerase chain reaction and Sanger sequencing. The sequenced region of both PDV-1 and PDV-2 contained partial genomes with motifs characteristic of pararetroviruses. BLAST analysis of PDV-1 and PDV-2 against the whole genome sequence of the black pepper showed integration of the PDV-1 at 22 loci in chromosome number 14, and PDV-2 at two loci in chromosome number 12 of black pepper. The integration was confirmed through amplification and sequencing of the junction regions. The present study suggests that both PDV-1 and PDV-2 occur as endogenous viruses in black pepper. Further studies are needed to determine whether these endogenous viruses occur in episomal forms, their complete genome sequence and whether they are activable under abiotic stress conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-021-00752-w.

A novel substituted derivative of sterol from marine actinomycetes Nocardiopsis alba MCCB 110 antagonistic to the aquaculture pathogen Vibrio harveyi.

In an attempt to screen antagonistic microorganisms from marine environment for the management of bacterial pathogens in aquaculture, an isolate of actinomycete MCCB 110 was segregated based on its comparatively higher inhibitory property on Vibrio harveyi (MCCB 111) and profound luminescent inhibition. Based on the culture characteristics, cell wall fatty acid profile and the nucleotide sequence of the 16S rRNA gene (1495 bp), the isolate was identified as Nocardiopsis alba. Solvent extraction of the fermentation broth followed by TLC and HPLC analyses resulted in the isolation of a major fraction active against luminescent Vibrio harveyi. Partial characterization of this bioactive fraction based on spectroscopic data obtained from FT-IR, UV, MS-MS and 1H NMR analyses identified it as a substituted derivative of sterol, and was recognized to differ from those reportedly produced by the same genus. The fraction was not toxic to VERO cell line and shrimp haemocytes up to 1000 ppm tested. The study demonstrated the potential of the putative probiotic Nocardiopsis alba (MCCB 110) and its novel extra-cellular bioactive product in the management of Vibrio harveyi in aquaculture.

Marine actinomycetes Nocardiopsis alba MCCB 110 has immunomodulatory property in the tiger shrimp Penaeus monodon.

Shrimp farming constitutes an important source of revenue and employment in many developing countries. However, the shrimp industry has always been plagued with infectious diseases having varied aetiologies. Dominated by non - specific immune mechanism, preventive health care strategy is the most appropriate approach to protect the crop. The present study evaluated the efficacy of an actinomycete, Nocardiopsis alba MCCB 110 in eliciting non - specific immune mechanism in Penaeus monodon having Vibrio harveyi as the challenge organism. Haemocyte count, total protein, phenoloxidase, reactive oxygen intermediates, acid and alkaline phosphatase as well as the gene expression of proPO, peroxinectin, transglutaminase, alpha 2-macroglobulin, astakine, crustin, and penaeidin-3 were evaluated. The results demonstrated that the phenoloxidase, respiratory burst, total protein, acid and alkaline phosphatases were higher in the haemolymph of shrimps fed with Nocardiopsis alba MCCB 110 incorporated feed before and after challenge with Vibrio harveyi, compared to those of placebo fed animals. Up-regulation of six immune genes (alpha 2 macroglobulin, penaeidin -3, transglutaminase, proPO, crustin and peroxinectin) during the post-challenge were recorded. Survival of shrimp among the Nocardiopsis alba administered ones was 83% while it was 50% in placebo fed group. The elevated levels of nonspecific immune gene transcripts and concurrent increase in non specific immunity besides the higher survival rate in the Nocardiopsis alba administered group demonstrated the immunomodulatory property of the marine actinomycete Nocardiopsis alba MCCB 110 in the tiger shrimp Penaeus monodon, and on administering it through diet shrimp could be protected from vibriosis especially of V. harveyi.

Effect of metals of treated electroplating industrial effluents on antioxidant defense system in the microalga Chlorella vulgaris.

The microalga Chlorella vulgaris is one of the prominent and most widely distributed green microalgae found in aquatic environments, often used in toxicity tests due to its sensitivity to various pollutants. To examine the toxicity of metals found in the effluent discharges from an electroplating industry, physicochemical parameters in the microalga C. vulgaris were measured. pH, turbidity, total dissolved solids, color, and the concentrations of metals such as chromium (1.97 mg/L), mercury (104.2 mg/L), and zinc (167.25 mg/L) were found exceeding the permissible limits. Several endpoints such as total protein content, reactive oxygen species (ROS) production, photosynthetic pigment contents, and antioxidant enzymatic activities, including those of superoxide dismutase (SOD) and catalase (CAT), were measured in C. vulgaris in response to treated electroplating industrial effluent (TEPIE). In addition, concentration-dependent morphological changes were also observed in response to TEPIE. Under both acute and chronic TEPIE exposure, increase in the ROS level was observed indicating increased production of ROS in C. vulgaris cells. The total protein and chlorophyll contents were found to be gradually decreasing in an effluent concentration-dependent manner. Moreover, lower concentrations of effluent stimulated the antioxidant enzyme systems. A concentration-dependent increase was observed in both SOD and CAT enzymatic activities. The results indicated toxic impairments by the effluent on the function of C. vulgaris in response to both acute and chronic exposure, indicating an urgent need of proper treatment processes/modification of the existing one of TEPIE, with continuous monitoring of the discharge of the pollutants into the aquatic ecosystems using biological assays.

Potential application of beta-1, 3 glucanase from an environmental isolate of Pseudomonas aeruginosa MCCB 123 in fungal DNA extraction.

Pseudomonas aeruginosa MCCB 123 was grown in a synthetic medium for beta-1,3 glucanase production. From the culture filtrate, beta-1,3 glucanase was purified with a molecular mass of 45 kDa. The enzyme was a metallozyme as its beta-1,3 glucanase activity got inhibited by the metal chelator EDTA. Optimum pH and temperature for beta-1,3 glucanase activity on laminarin was found to be 7 and 50 degrees C respectively. The MCCB 123 beta-1,3 glucanase was found to have good lytic action on a wide range of fungal isolates, and hence its application in fungal DNA extraction was evaluated. Beta-1,3 glucanase purified from the culture supernatant of P. aeruginosa MCCB 123 could be used for the extraction of fungal DNA without the addition of any other reagents generally used. Optimum pH and temperature of enzyme for fungal DNA extraction was found to be 7 and 65 degrees C respectively. This is the first report on beta-1,3 glucanase employed in fungal DNA extraction.

Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression.

Shrimp cell lines are yet to be reported and this restricts the prospects of investigating the associated viral pathogens, especially white spot syndrome virus (WSSV). In this context, development of primary cell cultures from lymphoid organs was standardized. Poly-l-lysine-coated culture vessels enhanced growth of lymphoid cells, while the application of vertebrate growth factors did not, except insulin-like growth factor-1 (IGF-1). Susceptibility of the lymphoid cells to WSSV was confirmed by immunofluoresence assay using monoclonal antibody against the 28 kDa envelope protein of WSSV. Expression of viral and immune-related genes in WSSV-infected lymphoid cultures could be demonstrated by RT-PCR. This emphasizes the utility of lymphoid primary cell culture as a platform for research in virus-cell interaction, virus morphogenesis, up and downregulation of shrimp immune-related genes, and also for the discovery of novel drugs to combat WSSV in shrimp culture.

Reducing Vibrio load in Artemia nauplii using antimicrobial photodynamic therapy: a promising strategy to reduce antibiotic application in shrimp larviculture.

We propose antimicrobial photodynamic therapy (aPDT) as an alternative strategy to reduce the use of antibiotics in shrimp larviculture systems. The growth of a multiple antibiotic resistant Vibrio harveyi strain was effectively controlled by treating the cells with Rose Bengal and photosensitizing for 30 min using a halogen lamp. This resulted in the death of >50% of the cells within the first 10 min of exposure and the 50% reduction in the cell wall integrity after 30 min could be attributed to the destruction of outer membrane protein of V. harveyi by reactive oxygen intermediates produced during the photosensitization. Further, mesocosm experiments with V. harveyi and Artemia nauplii demonstrated that in 30 min, the aPDT could kill 78.9% and 91.2% of heterotrophic bacterial and Vibrio population respectively. In conclusion, the study demonstrated that aPDT with its rapid action and as yet unreported resistance development possibilities could be a propitious strategy to reduce the use of antibiotics in shrimp larviculture systems and thereby, avoid their hazardous effects on human health and the ecosystem at large.

Application of primary haemocyte culture of Penaeus monodon in the assessment of cytotoxicity and genotoxicity of heavy metals and pesticides.

Lack of shrimp cell lines has hindered the study of pollutants which adversely affects shrimp health and its export value. In this context a primary haemocyte culture developed from Penaeus monodon was employed for assessing the cytotoxicity and genotoxicity of two heavy metal compounds, cadmium chloride and mercuric chloride and two organophosphate insecticides, malathion and monocrotophos. Using MTT assay 12 h IC(50) values calculated were 31.09 ± 16.27 µM and 5.52 ± 1.16 µM for cadmium chloride and mercuric chloride and 59.94 ± 52.30 mg l(-1) and 186.76 ± 77.00 mg l(-1) for malathion and monocrotophos respectively. Employing Comet assay, DNA damage inflicted by these pollutants on haemocytes were evaluated and the pollutants induced DNA damage in >60% of the cells. The study suggested that haemocyte culture could be used as a tool for quantifying cytotoxicity and genotoxicity of aquaculture drugs, management chemicals and pollutants.

Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays.

Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95 gl⁻¹), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 µg ml⁻¹ chloramphenicol, 100 µg ml⁻¹ streptomycin and 100 IU ml⁻¹ penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-2'-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24h. Susceptibility of the cells to WSSV was confirmed by immunofluorescence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT(50)/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC(50). The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals.

Pathological changes in Fenneropenaeus indicus experimentally infected with white spot virus and virus morphogenesis.

To demonstrate pathological changes due to white spot virus infection in Fenneropenaeus indicus, a batch of hatchery bred quarantined animals was experimentally infected with the virus. Organs such as gills, foregut, mid-gut, hindgut, nerve, eye, heart, ovary and integument were examined by light and electron microscopy. Histopathological analyses revealed changes hitherto not reported in F. indicus such as lesions to the internal folding of gut resulted in syncytial mass sloughed off into lumen, thickening of hepatopancreatic connective tissue with vacuolization of tubules and necrosis of rectal pads in hindgut. Virus replication was seen in the crystalline tract region of the compound eye and eosinophilic granules infiltrated from its base. In the gill arch, dilation and disintegration of median blood vessel was observed. In the nervous tissues, encapsulation and subsequent atrophy of hypertrophied nuclei of the neurosecretory cells were found. Transmission electron microscopy showed viral replication and morphogenesis in cells of infected tissue. De novo formed vesicles covered the capsid forming a bilayered envelop opened at one end inside the virogenic stroma. Circular vesicles containing nuclear material was found fused with the envelop. Subsequent thickening of the envelop resulted in the fully formed virus. In this study, a correlation was observed between the stages of viral multiplication and the corresponding pathological changes in the cells during the WSV infection. Accordingly, gill and foregut tissues were found highly infected during the onset of clinical signs itself, and are proposed to be used as the tissues for routine disease diagnosis.

Copper-induced changes in tissue enzyme activity in a freshwater mussel.

Changes in enzyme activity levels are of great diagnostic value. Lysosomal membrane is often the target of injury by xenobiotics, resulting in destabilization. Variations in the activity of acid phosphatase (ACP) a marker enzyme, in gills and hepatopancreas of the freshwater mussel Lamellidens corrianus (Lea) exposed to different concentrations of copper for 24, 120, and 168 h are discussed. The aim was to determine if the metal caused any variation in enzyme activity in the two tissues studied and, if so, whether the length of exposure had any influence on enzyme activity. ACP activity was determined as described in Sigma Technical Bulletin No. 104 and expressed as micromoles of p-nitrophenol liberated per milligram of protein per hour. Both concentration of the metal and length of exposure were found to influence enzyme activity. Higher concentrations of metals are assumed to induce stress proteins like metallothioneins.

Involvement of spicules in the formation of vaccinia virus envelopes elucidated by a conditional lethal mutant.

The envelope of immature vaccinia virions consists of a lipoprotein bilayer upon which a precise curvature is imposed by acquisition of an external scaffold of spicules. Self-assembly of this tegument was examined employing our ts 6757 mutant, which induces accumulation of immature envelopes at the restrictive temperature. With ts 6757 the envelope bilayers were also assembled into an alternative membrane configuration in the form of flexible cylinders or tubes of uniform width, lacking the spicule coat. Such tubes became extensions of or were continuous with the spherical virion envelopes. The approximately 65 kDa spicule protein, L65, product of gene D13L on the HindIII map, generally designated as a late protein, was expressed as an early function in presence of hydroxyurea, an inhibitor which entirely blocked vaccinia DNA synthesis without stopping assembly of immature envelopes. Labeling of thin sections by immunogold for electron microscopy demonstrated that L65 is present at the surface of immature virions, consistent with the position of spicules on envelopes. Transiency of the spicule scaffold was documented by (a) absence of L65 from intracellular mature virions (IMV) and (b) rapid turnover of L65 during ts 6757 virus replication at the permissive temperature but conservation of this protein at restrictive temperature, as demonstrated in pulse-chase experiments. Time-related decrease in MW of L65 to a smaller polypeptide is interpreted as evidence suggesting that the spicules attached to the envelope are assembled from a higher MW precursor.

Organization of the vaccinia envelope and relationship to the structure of intracellular mature virions.

Protrusions covering the surface of intracellular mature virions (IMV), termed by us surface tubular elements (STE), are released in a quasi-intact form during stripping of the envelope. The concentrated, reproducibly isolable STE were shown to contain the 58-kDa 4c polypeptide and prominent protein antigens residing at the surface of IMV. The major core protein 4b, identified as a minor contaminant of STE, presumably became detached along with STE during the shearing off process. Antibodies against protein 4b became specifically bound to the surface of isolated cores, where a palisade layer of rodlets occurs. The same antibodies absorbed onto isolated STE where similar rodlets were evident. Based on the new observations we constructed a model of the organization of the IMV envelope and its relationship to the core.

Vaccinia virion surface polypeptide Ag35 expressed from a baculovirus vector is targeted to analogous poxvirus and insect virus components.

Polypeptide Ag35, a major early component of the vaccinia surface, is integrated into the formative viral lipoprotein tegument. To ascertain whether positioning of Ag35 is due to its general affinity for newly assembled viral membranes we created a recombinant A12 vector to express the vaccinia protein. The baculovirus system was chosen because intranuclear virions of this agent are likewise enclosed inside newly formed envelopes. Comparable infections of two insect cell lines established that more abundant synthesis occurred in High Five (H5) than in SF9 cells. We, therefore, used H5 cells for most experiments reported here. Combined analyses by PAGE, Western blotting, and immunocytology, using light and electron microscopy, revealed a dissemination of Ag35 throughout the cell. Higher concentrations were evident at the cell surface, nuclear perimeter, and within intranuclear virogenic stroma. The association with the virogenic stroma was of specific interest with respect to vaccinia development because it showed a similarity in the targeting of Ag35 toward intranuclear DNA-protein foci of baculovirus which are analogous to the vaccinia-specified cytoplasmic "factories." A further remarkable analogy concerns association of Ag35 with intranuclear baculovirus envelopes, revealing a propensity of Ag35 for nascent viral lipoprotein membranes.

Separation of Lymnaea stagnalis hemocytes by density gradient centrifugation.

A chelating anti-clumping (alpha-C) buffer allowed blood cells (hemocytes) of a gastropod, Lymnaea stagnalis to be separated by discontinuous Percoll density gradient centrifugation. The hemocytes of L. stagnalis were separated into five fractions, having a density lower than 10, 20, 30, 40, and 50% Percoll, respectively. Trypan blue exclusion assays showed viability of separated hemocytes to be between 81 and 89%. Cytospin preparations of these hemocytes were examined. Small cells were mainly observed at high densities; at lower densities medium and large hemocytes were also present. No absolute separation was achieved. Some density fractions were enriched for hemocytes with regard to the distributions of two endogenous lysosomal enzymes (alpha-naphthyl acetate esterase and acid phosphatase).

A prominent antigenic surface polypeptide involved in the biogenesis and function of the vaccinia virus envelope.

Polypeptides of the vaccinia virus envelope exposed on the surface were identified by means of sulfo-N-hydroxysuccinimidobiotin as a surface tag. Among surface expressed polypeptides is the 35-kDa antigen, previously designated Ag35. Both monoclonal (mAb) and monospecific affinity pure antibodies directed against Ag35 neutralized vaccinia infectiousness, indicating that this prominent surface antigen has a function during early virus-host cell interactions. The binding of several monoclonal antibodies to various regions of Ag35 was tested by reacting CNBr fragments, derived from the polypeptide, employing Western blotting. All mAbs tested reacted with the same region of Ag35. Estimation of the molecular weights (MW), based on migration of the CNBr peptides in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that those partial digestion products which contained a proline-rich 99 amino acid limit digest fragment were present at a position approximately 12.5 kDa larger than that predicted from the DNA sequence. By contrast, partial and limit digest products lacking the proline-rich fragment migrated to the MW position expected from the length of the DNA sequence. This observation demonstrates that departure from a predicted 22.3 kDa to an anomalous MW of Ag35 is conferred by the proline-rich peptide. The surface location of Ag35 was confirmed by immune electron microscopy. In a competition test the binding specificity of mAb and affinity-purified antibodies at the surface of virions could be demonstrated. Evidence for an association of Ag35 with the virus envelope at various stages during biogenesis of vaccinia was obtained by immune electron microscopy of whole mounts and thin sections. Presence of Ag35 as an early component of immature and mature virions, probably residing in the bilayer membrane structure was detected. A distinction can, therefore, be made between Ag35 and several other vaccinia envelope polypeptides which are synthesized as late functions and added during late stages of envelope assembly.

Number and types of hemocytes in Sunetta scripta and Villorita cyprinoides var. cochinensis (Bivalvia), and leukocytosis subsequent to bacterial challenge.

The number and types of hemocytes in four size groups of the clam species Sunetta scripta and Villorita cyprinoides var. cochinensis, and leukocytosis in the 38- to 40-mm-size-group clams of both species subsequent to challenge with Vibrio alginolyticus at a concentration of 1 x 10(8) cells/0.02 ml of sterile 2% saline was investigated. In S. scripta, the mean total hemocyte count in the 42- to 44-mm size group was significantly lower than that of the three other size groups but there was no significant variation in total cell counts in the four size groups of V. cyprinoides var. cochinensis. Only two types of hemocytes, granulocytes and agranulocytes, occur and the percentage of agranulocytes was roughly half of that of granulocytes. The data on the effects of sham injection and Vibrio injection suggest that there is significant leukocytosis early in both clam species as a result of sham injection; the bacterial challenge produces significant leukocytosis in V. cyprinoides var. cochinensis both early (6 and 12 hr) and later (48,96 and 120 hr), but only at 48 hr in S. scripta; and in both clam species there is significant increase in total cell counts in Vibrio-injected ones than in untampered controls at various time intervals.