Propagation of IHHNV (Infectious hypodermal and haematopoietic necrosis virus)/PstDVI and MBV (Monodon baculovirus)/PmNV in shrimp primary haemocyte culture.

In vitro propagation of shrimp viruses IHHNV (Infectious hematopoietic hypodermal necrosis virus)/PstDV1 and MBV (Monodon baculovirus)/PmNV in the primary haemocyte culture was successfully be accomplished in the present study. The haemocytes were maintained in double-strength L15 medium with growth supplements and antibiotics. Cytopathic changes observed in the cells were detachment, slendering of cells, vacuolation, and rounding of the cells. Replication of viruses in the culture was confirmed through the expression of DNA polymerase, polyhedrin and lef 4 of MBV and capsid gene of IHHNV. Ultrathin sections of haemocytes observed through electron microscopy revealed scattered chromatin, virogenic stroma and presence of viruses and occlusion bodies (MBV). The study revealed that primary haemocyte culture could be used as a platform for the propagation of a variety of shrimp viruses.

Exploring bacterial diversity in Arctic fjord sediments: a 16S rRNA-based metabarcoding portrait.

The frosty polar environment houses diverse habitats mostly driven by psychrophilic and psychrotolerant microbes. Along with traditional cultivation methods, next-generation sequencing technologies have become common for exploring microbial communities from various extreme environments. Investigations on glaciers, ice sheets, ponds, lakes, etc. have revealed the existence of numerous microorganisms while details of microbial communities in the Arctic fjords remain incomplete. The current study focuses on understanding the bacterial diversity in two Arctic fjord sediments employing the 16S rRNA gene metabarcoding and its comparison with previous studies from various Arctic habitats. The study revealed that Proteobacteria was the dominant phylum from both the fjord samples followed by Bacteroidetes, Planctomycetes, Firmicutes, Actinobacteria, Cyanobacteria, Chloroflexi and Chlamydiae. A significant proportion of unclassified reads derived from bacteria was also detected. Psychrobacter, Pseudomonas, Acinetobacter, Aeromonas, Photobacterium, Flavobacterium, Gramella and Shewanella were the major genera in both the fjord sediments. The above findings were confirmed by the comparative analysis of fjord metadata with the previously reported (secondary metadata) Arctic samples. This study demonstrated the potential of 16S rRNA gene metabarcoding in resolving bacterial composition and diversity thereby providing new in situ insights into Arctic fjord systems.

Antibacterial activity and modes of action of a novel hepcidin isoform from the shrimp scad, Alepes djedaba (Forsskål, 1775).

Hepcidin, initially identified in human blood ultrafiltrate as cysteine rich Liver Expressed Antimicrobial Peptide (LEAP-1), is a core molecular conduit between iron trafficking and immune response. Though a great share of studies has been focused on the iron regulatory function of hepcidins, investigations on the antimicrobial aspects are relatively less. The present study is aimed at identification of hepcidin from a teleost fish, Alepes djedaba followed by its recombinant expression, testing antibacterial property, stability and evaluation of cytotoxicity. Modes of action on bacterial pathogens were also examined. A novel hepcidin isoform, Ad-Hep belonging to the HAMP1 (Hepcidin antimicrobial peptide 1) group of hepcidins was identified from the shrimp scad, Alepes djedaba. Ad-Hep with 2.9 kDa size was found to be a cysteine rich, cationic peptide (+4) with antiparallel beta sheet conformation, a furin cleavage site (RXXR) and 'ATCUN' motif. It was heterologously expressed in E. coli Rosettagami B(DE3)PLysS cells and the recombinant peptide, rAd-Hep was found to have significant antibacterial activity, especially against Edwardsiella tarda, Vibrio parahaemolyticus and Escherichia coli. Membrane depolarization followed by membrane permeabilization and Reactive Oxygen Species (ROS) production were found to be the modes of action of rAd-Hep on bacterial cells. Ad-Hep was found to be non-haemolytic to hRBC and non-cytotoxic in mammalian cell line. Stability of the peptide at varying temperature, pH and metal salts qualify them for applications in vivo. With significant bactericidal activity coupled with direct killing mechanisms, the rAd-Hep can be a promising drug candidate for therapeutic applications in medicine and fish culture systems.

Marine actinomycete Streptomyces variabilis S26 as a biocontrol agent for vibriosis in shrimp larval rearing systems.

Indiscriminate use of antibiotics has led to the emergence of antibiotic-resistant microbes and the loss of natural flora in aquaculture systems necessitating the ban of many of these chemotherapeutants in aquaculture. Actinobacteria play a profound role in the biogeochemical cycling in the marine environment and represent the principal source of secondary metabolites with antimicrobial property. In the present study, 98 marine-derived actinomycete isolates were screened for antimicrobial activity against the common aquatic pathogens. A potent actinomycete isolate S26, identified as Streptomyces variabilis based on 16 S ribosomal RNA (rRNA) gene sequencing was then checked for the production of antibiotic in five different fermentation media and the one which showed maximum production was chosen for further study. Optimization of the fermentation medium for secondary metabolite production was carried out by response surface methodology (RSM) using DESIGN EXPERT. The analysis of variance (ANOVA) of the quadratic regression model demonstrated that the model was highly significant for the response concerned that is, antimicrobial activity as evident from the Fisher's F- test with a very low probability value [(P model>F) = 0.0001]. Of the 10 different solutions suggested by the software, the most suitable composition was found to be starch, 1.38%; soy powder, 0.88%; ammonium sulfate, 0.16% and salinity, 27.76‰. S. variabilis S26 cultured in the optimized production medium was applied in the Penaeus monodon larval rearing system and the total Vibrio count and survival rate were estimated. S. variabilis S26 treatment showed a significant reduction in vibrios and conferred better protection to P. monodon in culture system compared with control.

Mangrove-associated endomycota: diversity and functional significance as a source of novel drug leads.

Endophytic fungi are known for their unprecedented ability to produce novel lead compounds of clinical and pharmaceutical importance. This review focuses on the unexplored fungal diversity associated with mangroves, emphasizing their biodiversity, distribution, and methodological approaches targeting isolation, and identification. Also highlights the bioactive compounds reported from the mangrove fungal endophytes. The compounds are categorized according to their reported biological activities including antimicrobial, antioxidant and cytotoxic property. In addition, protein kinase, α-glucosidase, acetylcholinesterase, tyrosinase inhibition, antiangiogenic, DNA-binding affinity, and calcium/potassium channel blocking activity are also reported. Exploration of these endophytes as a source of pharmacologically important compounds will be highly promising in the wake of emerging antibiotic resistance among pathogens. Thus, the aim of this review is to present a detailed report of mangrove derived endophytic fungi and to open an avenue for researchers to discover the possibilities of exploring these hidden mycota in developing novel drug leads.

Ambient copper modulates immunocompetence and induces physiological responses in Penaeus monodon against white spot syndrome virus infection.

Among trace metals, copper is essential for crustaceans' normal growth and metabolism. In the present study, an attempt was made to determine whether the addition of copper in rearing water influences the physiological and immunological responses of Penaeus monodon to white spot syndrome virus infection (WSSV). Adult P. monodon were distributed in experimental tanks and exposed to 0, 0.05, 0.1, 0.2 and 0.3 mg l-1 copper concentrations. After 14 days, the shrimps were challenged with WSSV and the biochemical/immune variables were determined on post-metal exposure day 14 and post-challenge days 2 and 5. Significant variations could be observed in the haemolymph (biochemical and immune) variables of P. monodon on exposure to copper and WSSV challenge. Shrimps exposed to copper at 0.1 mg l-1 showed higher total haemocyte count, phenol oxidase activity, nitro blue tetrazolium salt reduction, alkaline/acid phosphatase activity, total protein, carbohydrates, lipids, glucose and cholesterol besides maximum post-challenge survival. However, exposure to copper at 0.2 and 0.3 mgl-1 increased the susceptibility to WSSV infection, showing a decrease in the biochemical/immune variables. Therefore, the present study concludes that copper in ambient water induces immunomodulation and evokes physiological responses in P. monodon at sub-lethal doses. Immunostimulatory effects elicited by copper at 0.1 mg l-1 enhanced the immunocompetence and reduced the susceptibility of P. monodon to WSSV infection, conferring protection to the animals and resulting in higher survival.

In vitro propagation of infectious hypodermal hematopoietic necrosis virus (Penaeus stylirostris penstyldensovirus) in PmLyO-Sf9 cells.

Infectious hypodermal hematopoietic necrosis virus (IHHNV/PstDVI) was isolated and propagated in the hybrid shrimp-insect cell line PmLyO-Sf9. A few hours after inoculation with an infected tissue extract or virus suspension, cytopathic changes could be observed in the cell line, including clustering, enlargement, syncytium formation, granulation, vacuole formation, tapering, irregularities in the plasma membrane with extended tails, detachment, cell death, and accumulation of cellular debris. Expression of viral genes, the presence of virions, and cytological changes observed using transmission electron microscopy suggested replication of the virus in these cells. The virus was purified by ultracentrifugation, negatively stained, and examined using an electron microscope, and the purified virus was found to be infectious both in vitro and in vivo. This development opens avenues for the study of the basic molecular mechanism of IHHNV infection, pathogenesis, and replication, which is much needed for developing an antiviral strategy in aquaculture.

A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression.

BACKGROUND: Invertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides. RESULTS: This study identified and characterized a novel homolog of ALF (Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four ß sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs. CONCLUSION: Antibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.

Characterisation of a novel crustin isoform from mud crab, Scylla serrata (Forsskål, 1775) and its functional analysis in silico.

A 336-base pair (bp) sized mRNA sequence encoding 111 amino acid size crustin isoform (MC-crustin) was obtained from the gill sample of the green mud crab, Scylla serrata. MC-crustin possessed an N-terminal signal peptide region comprising of 21 amino acid residues, followed by a 90 amino acid mature peptide region having a molecular weight of 10.164 kDa, charge + 4.25 and theoretical pI of 8.27. Sequence alignment and phylogenetic tree analyses revealed the peptide to be a Type I crustin, with four conserved cysteine residues forming the cysteine rich region, followed by WAP domain. MC-crustin was cationic with cysteine/proline rich structure and was predicted with antimicrobial, anti-inflammatory, anti-angiogenic and anti-hypertensive property making it a potential molecule for possible therapeutic applications.

Functional characterization of a histone H2A derived antimicrobial peptide HARRIOTTIN-1 from sicklefin chimaera, Neoharriotta pinnata.

Antimicrobial peptides (AMPs) are gene encoded short peptides which play an important role in the innate immunity of almost all living organisms ranging from bacteria to mammals. Histones play a very important role in defense as precursors to bioactive peptides. The present study is an attempt to decipher the antimicrobial activity of a histone H2A derived peptide, Harriottin-1 from sicklefin chimaera, Neoharriotta pinnata. Analysis in silico predicted the molecule with potent antibacterial and anticancer property. The Harriottin-1 was recombinantly produced and the recombinant peptide rHar-1 demonstrated potent antibacterial activity at 25 µM besides anticancer activity. The study strongly suggests the importance of histone H2A derived peptides as a model for the design and synthesis of potent peptide drugs.

A hepatic antimicrobial peptide, hepcidin from Indian major carp, Catla catla: molecular identification and functional characterization.

BACKGROUND: Increase of antibiotic resistance in pathogenic microbes necessitated novel molecules for curing infection. Antimicrobial peptides (AMPs) are the gene-encoded evolutionarily conserved small molecules with therapeutic value. AMPs are considered as an alternative drug for conventional antibiotics. Hepcidin, the cysteine-rich antimicrobial peptide, is an important component in innate immune response. In this study, we identified and characterized hepcidin gene from the fish, Catla catla (Indian major carp) and termed it as Cc-Hep. RESULTS: Open reading frame of Cc-Hep consists of 261 base pair that encodes 87 amino acids. Cc-Hep is synthesized as a prepropeptide consisting of 24 amino acid signal peptide, 36 amino acid propeptide, and 26 amino acid mature peptide. Sequence analysis revealed that Cc-Hep shared sequence similarity with hepcidin from Sorsogona tuberculata. Phylogenetic analysis indicated that Cc-Hep was grouped with HAMP2 family. Structure analysis of mature Cc-Hep identified two antiparallel beta sheets stabilized by four disulphide bonds and a random coil. The mature peptide region of Cc-Hep has a charge of + 2, isoelectric value 8.23 and molecular weight 2.73 kDa. CONCLUSION: Functional characterization predicted antibacterial, antioxidant, and anticancer potential of Cc-Hep, which can be explored in aquaculture or human health care.

A Novel Approach of Transducing Recombinant Baculovirus into Primary Lymphoid Cells of Penaeus monodon for Developing Continuous Cell Line.

Cell line development from shrimp is not a novel venture as researchers across the globe have been trying to have crustacean cell lines over 30 years. The reason for not attaining a crustacean or precisely a shrimp cell line is believed to be the replicative senescence and the inability to maintain telomere length in vitro. Moreover, spontaneous in vitro transformations do not happen in shrimp cells. Oncogenic induction in primary cell culture is one of the ways to attain in vitro transformation by way of disrupting the mechanisms which involve cellular senescence. In this context, a recombinant baculovirus with shrimp viral promoter IHHNV-P2 was used for the transduction aimed at immortalization. An oncogene, H-ras, was successfully amplified and cloned in to the baculoviral vector, downstream to shrimp viral promoter IHHNV-P2 and upstream to GFP. Recombinant baculovirus with H-ras was generated and used for transduction into shrimp lymphoid cells during early dividing stage. Accordingly, fibroblast-like primary cell culture got developed, and H-ras and GFP expression could be confirmed. The study suggests that the simple method of incubating recombinant baculovirus with minced tissue enables in vitro transduction during early dividing stage of the cells, and the transduction efficiency gets enhanced by adding 5 mM sodium butyrate to the culture medium.

ß-Defensins from common goby (Pomatoschistus microps) and silver trevally (Pseudocaranx georgianus): Molecular characterization and phylogenetic analysis.

Antimicrobial peptides (AMPs) are biologically active molecules involved in host defense present in a variety of organisms. They are an integral component of innate immunity, forming a front line of defense against potential pathogens, including antibiotic-resistant ones. Fishes are proven to be a prospective source of AMPs as they are constantly being challenged by a variety of pathogens and the AMPs are reported to play an inevitable role in fish immunity. Among them, ß-defensins form one of the most studied multifunctional peptides with early evolutionary history and recently being considered as host defense peptides. The present study highlights the first-ever report on ß-defensin AMP sequences from common goby (Pomatoschistus microps) and silver trevally (Pseudocaranx georgianus). A 192 bp cDNA fragment with an open reading frame encoding 63 amino acids (aa) comprising a 20 aa signal peptide region at the N-terminal was obtained from the mRNA of gill tissue of both P. microps and P. georgianus by RT-PCR. These peptide sequences when characterized in silico at the molecular level revealed a 43 aa cationic mature peptide with the signature intra-molecular disulphide bonded cysteine residue pattern ascertaining its ß-defensin identity, further confirmed by phylogenetic analysis. The data collected will pave the way for further research on varied facets of the peptide-like, tissue level expressions, antimicrobial activities on commonly encountered pathogens, and its feasibility as a therapeutant in the aquaculture scenario.

ß-Defensin from the Asian Sea Bass, Lates calcarifer: Molecular Prediction and Phylogenetic Analysis.

Antimicrobial peptides (AMPs) are an important element of the innate immune system of all living organisms and serve as a barrier that safeguards the organisms against a wide range of pathogens. Fishes are proven to be a prospective source of AMPs, and ß-defensins form an important family of AMPs with potent antimicrobial, chemotactic and immunomodulatory activities. The present study reports a ß-defensin AMP sequence (Lc-BD) from the Asian sea bass, Lates calcarifer, a commercially important fish species in tropical and subtropical regions of Asia and the Pacific. A 202-bp cDNA fragment with an open reading frame encoding 63 amino acids (aa) was obtained from the mRNA of gill tissue by RT-PCR. The deduced aa sequence of Lc-BD possessed a signal and a mature peptide region with 20 and 43 aa residues, respectively. Lc-BD was characterized at the molecular level, and a molecular weight of 5.24 kDa and a net charge of +4.5 was predicted for the mature peptide. The molecular characterization of Lc-BD revealed the presence of three intramolecular disulphide bonds involving the six conserved cysteine residues in the sequence, and the phylogenetic analysis of Lc-BD showed a close relationship with ß-defensins from fishes like Siniperca chuatsi, Argyrosomus regius, Trachinotus ovatus and Oplegnathus fasciatus.

Molecular characterization of a novel ß-defensin isoform from the red-toothed trigger fish, Odonus niger (Ruppel, 1836).

BACKGROUND: The concern regarding a post-antibiotic era with increasing drug resistance by pathogens imposes the need to discover alternatives for existing antibiotics. Antimicrobial peptides (AMPs) with their versatile therapeutic properties are a group of promising molecules with curative potentials. These evolutionarily conserved molecules play important roles in the innate immune system of several organisms. The ß-defensins are a group of cysteine rich cationic antimicrobial peptides that play an important role in the innate immune system by their antimicrobial activity against the invading pathogens. The present study deals with a novel ß-defensin isoform from the red-toothed trigger fish, Odonus niger. Total RNA was isolated from the gills, cDNA was synthesized and the ß-defensin isoform obtained by polymerase chain reaction was cloned and subjected to structural and functional characterization in silico. RESULTS: A ß-defensin isoform could be detected from the gill mRNA of red-toothed trigger fish, Odonus niger. The cDNA encoded a 63 amino acid peptide, ß-defensin, with a 20 amino acid signal sequence followed by 43 amino acid cationic mature peptide (On-Def) having a molecular weight of 5.214 kDa and theoretical pI of 8.89. On-Def possessed six highly conserved cysteine residues forming disulfide bonds between C1-C5, C2-C4, and C3-C6, typical of ß-defensins. An anionic pro-region was observed prior to the ß-defensin domain within the mature peptide. Clustal alignment and phylogenetic analyses revealed On-Def as a group 2 ß-defensin. Furthermore, it shared some structural similarities and functional motifs with ß-defensins from other organisms. On-Def was predicted to be non-hemolytic with anti-bacterial, anti-viral, anti-fungal, anti-cancer, and immunomodulatory potential. CONCLUSION: On-Def is the first report of a ß-defensin from the red-toothed trigger fish, Odonus niger. The antimicrobial profile showed the potential for further studies as a suitable candidate for antimicrobial peptide therapeutics.

Immortalization of shrimp lymphoid cells by hybridizing with the continuous cell line Sf9 leading to the development of 'PmLyO-Sf9 '.

Shrimp progressively gets more attention among marine invertebrates from researchers all over the world due to it being a healthy food as well as having economic importance. There were a lot of attempts to develop a continuous cell line from shrimp but none successful. In this context a novel hybrid cell line named 'PmLyO-Sf9' could be developed by fusing shrimp lymphoid organ cells with Sf9 cells after to metabolic blocking of Sf9 cells using puromycin and actinomycin D and effecting the fusion by way of PEG application. The cells are maintained and multiplied in a mixture of SCCM and TNM-FH having osmolality 550 mOsm kg-1 and pH 6.8. Transmission electron microscopy of the hybrid cells revealed the presence of two nuclei during the initial stages and a single nucleus subsequently. The cell line is with shrimp and Sf9 genomic components and shrimp specific protein and is susceptible to WSSV. Shrimp elongation factor, Sf9 beta-actin, shrimp STAT and peroxinectin could be expresses through RT-PCR in the cell line. This is the first successful report of a hybrid cell line with shrimp genomic components and envisaged to be recognized a model system for multitudes of biomedical research in vitro. The cell line is in the National Cell Line Repository of ICAR - National Bureaue of Fish Genetic Resources, Lucknow, India.

Molecular and Functional Characterization of an Anti-lipopolysaccharide Factor Mm-ALF from Speckled Shrimp Metapenaeus monoceros.

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides of approximately 100 amino acid residues with a broad spectrum of antimicrobial activity. It is an amphipathic peptide with an N-terminal hydrophobic region and a lipopolysaccharide binding domain (LBD). In the present study, we report an isoform of the anti-lipopolysaccharide factor (Mm-ALF) from the speckled shrimp, Metapenaeus monoceros. A 359 bp cDNA encoded 119 amino acids, and the sequence showed 99.16% similarity to ALF from the shrimp Fenneropenaeus indicus. The mature peptide of 94 amino acids has a net charge of +8, molecular weight 10.62 kDa, and pI 10.11. The mature peptide Mm-ALF was recombinantly expressed in E. coli Rosetta-gami cells, and the peptide was isolated and purified. The rMm-ALF exhibited notable antibacterial activity against Gram-positive (Staphylococcus aureus and Bacillus cereus) and Gram-negative (Escherichia coli, Edwardsiella tarda, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio proteolyticus, Vibrio cholerae and Vibrio fluvialis) bacteria.

Biocompatibility of polyhydroxybutyrate-co-hydroxyvalerate films generated from Bacillus cereus MCCB 281 for medical applications.

Polyhydroxyalkanoates (PHAs) are natural polyesters produced by microorganisms as a source of intracellular energy reserves. These polymers have been extensively studied for tissue engineering and drug delivery applications due to their desirable material properties. Solvent-cast film of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), produced by Bacillus cereus MCCB 281 was characterized to study the surface morphology, roughness, thermal and mechanical properties. PHBV films were slightly hydrophilic with an average surface roughness of 43.66 nm. In vitro cell viability and proliferation studies on PHBV film surface investigated using L929 fibroblasts showed good cell attachment and proliferation. Hemocompatibility of PHBV evaluated by hemolysis assay, in vitro platelet adhesion and coagulation assays demonstrated good blood compatibility for use as blood contact graft materials. Therefore, PHBV produced from the marine bacterium favoured cellular growth of L929 fibroblasts indicating its potential to be used as a biomaterial substrate for cell adhesion in tissue engineering and medical applications.

'PmLyO-Sf9 - WSSV complex' could be a platform for elucidating the mechanism of viral entry, cellular apoptosis and replication impediments.

White spot syndrome virus (WSSV) is the most devastating pathogen found in shrimp aquaculture. The lack of certified continuous/established cell lines from penaeid shrimp restricts in vitro studies on the viruses to bring out effective prophylactic and therapeutic measures. In this context, a novel hybrid cell line named, PmLyO-Sf9, consisting of shrimp and Sf9 genomes has been established and employed to study WSSV susceptibility and multiplication. The hybrid cells were exposed to the shrimp virus WSSV and cytopathic effects (CPE) such as (a) enlargement of cells, (b) cessation cell division, (c) granulation of cytoplasm, (d) rounding off of cells, shortening and disappearance of tail-like structures and (e) detachment from the flask. Expression of immediate early genes such as ie 1, dnapol, rr1, tk-tmk, and pk 1could be confirmed indicating that viral DNA replication in the PmLyO-Sf9 took place followed by the expression of late genes such as VP-28, VP-26, VP-15 and VP-19. Electron micrograph of WSSV infected cells demonstrated marginated dense zones in the nucleus with clumped chromatin, and the mid zone with virus-like particles. However, neither discrete virus particles nor the culture supernatant having infectivity could be observed suggesting that virions were not getting formed in the cells. This is the first report of the susceptibility of PmLyO-Sf9 to WSSV, and the 'PmLyO-Sf9 - WSSV Complex' formed, defined as the infected status of PmLyO-Sf9 with WSSV, could be of use for unraveling at molecular level the mechanism of viral entry, replication impediments and cellular apoptosis.

Anti-lipopolysaccharide Factor from Crucifix Crab Charybdis feriatus, Cf-ALF2: Molecular Cloning and Functional Characterization of the Recombinant Peptide.

Antilipopolysaccharide factors (ALFs) are important effectors of innate immunity in crustaceans with broad spectrum antimicrobial activity. Present study deals with the molecular and functional characterization of a 98-amino acid ALF isoform from, crucifix crab, Charybdis feriatus termed as Cf-ALF2. The ALF isoform Cf-ALF2 exhibits characteristic features of an AMP including a cationic net charge of + 9 and a total hydrophobic ratio of 34%. Recombinant peptide rCf-ALF2 showed remarkable antimicrobial activity against Gram-negative and Gram-positive bacteria especially against Staphylococcus aureus (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 5 µM) and Escherichia coli (MIC 10 µM and MBC 20 µM). Using scanning electron microscopy, bacterial membrane blebbing, disruption, and cell content leakage were observed in peptide treated E. coli. The recombinant peptide was found to be non-hemolytic and non-cytotoxic in NCI-H460 cell line at the highest tested concentration (20 µM). Thus, this study identified a novel isoform of ALF from C. feriatus and revealed the potent antimicrobial property of the recombinant peptide Cf-ALF2 and the future prospects of using the peptide for therapeutic applications in the future.