Evaluation of Chitosan Derivatives Modified Mesoporous Silica Nanoparticles as Delivery Carrier.

Chitosan is a non-toxic biological material, but chitosan is insoluble in water, which hinders the development and utilization of chitosan. Chitosan derivatives N-2-Hydroxypropyl trimethyl ammonium chloride (N-2-HACC) and carboxymethyl chitosan (CMCS) with good water solubility were synthesized by our laboratory. In this study, we synthesized mesoporous SiO2 nanoparticles by the emulsion, and then the mesoporous SiO2 nanoparticles were modified with γ-aminopropyltriethoxysilane to synthesize aminated mesoporous SiO2 nanoparticles; CMCS and N-2-HACC was used to cross-link the aminated mesoporous SiO2 nanoparticles to construct SiO2@CMCS-N-2-HACC nanoparticles. Because the aminated mesoporous SiO2 nanoparticles with positively charged can react with the mucous membranes, the virus enters the body mainly through mucous membranes, so Newcastle disease virus (NDV) was selected as the model drug to evaluate the performance of the SiO2@CMCS-N-2-HACC nanoparticles. We prepared the SiO2@CMCS-N-2-HACC nanoparticles loaded with inactivated NDV (NDV/SiO2@CMCS-N-2-HACC). The SiO2@CMCS-N-2-HACC nanoparticles as delivery carrier had high loading capacity, low cytotoxicity, good acid resistance and bile resistance and enteric solubility, and the structure of NDV protein encapsulated in the nano vaccine was not destroyed. In addition, the SiO2@CMCS-N-2-HACC nanoparticles could sustain slowly released NDV. Therefore, the SiO2@CMCS-N-2-HACC nanoparticles have the potential to be served as delivery vehicle for vaccine and/or drug.

The improved antiviral activities of amino-modified chitosan derivatives on Newcastle virus.

Chitosan is widely used as a medical material because of its excellent biological activities. However, the low solubility of natural chitosan limited its medicinal activity to some extent. The solubility can be improved by introducing more active groups and lowering molecular weight. Therefore, 6-amine chitosan derivatives were synthesized in this paper since more active groups were introduced to increase the medicinal activity. Those derivatives were characterized by elemental analysis, HPLC, and FT-IR and the antiviral activity was tested by hemagglutination tests. Finally, 6-amine chitosan derivatives improved the antiviral activity, especially after the introduction of bromine ion. When 6-deoxy-6-bromo-N-phthaloyl chitosan was 1 g/L, they reduced the hemagglutination titer of virus to zero. The RT-PCR result showed that the expression level of TNF-α and IFN-ß increased significantly, which indicated that the antiviral activity of amino-modified chitosan worked through the stimulation of immune response.

Lithium chloride functions as Newcastle disease virus-induced ER-stress modulator and confers anti-viral effect.

Newcastle disease is a severe clinical manifestation of avian species caused by Newcastle disease virus (NDV). Although several vaccination strategies are available to protect poultry against NDV infection, even then, outbreaks have been reported in the vaccinated birds. The lack of therapeutics against NDV makes the need for effective anti-viral drugs is of utmost importance. Lithium Chloride (LiCl) is a widely prescribed drug for the treatment of bipolar disorder, acute brain injuries, and chronic neurodegenerative diseases. Also, LiCl has been repurposed as an effective anti-viral drug for some viral infections. In the present work, we have investigated the efficacy of LiCl to inhibit NDV replication using in vitro, in ovo, and in vivo models. Our results collectively showed the modulation of NDV replication after the LiCl treatment. We also demonstrated that NDV induces endoplasmic reticulum stress (ER-stress), and a stress-inducible ER chaperone, glucose-regulating protein 78 (GRP78), was found to be over-expressed after NDV infection. Subsequently, the treatment of NDV infected cells with LiCl significantly reduced the transcript and protein levels of GRP78. Finally, we concluded that LiCl treatment protects the cells from ER-stress induced by the NDV infection.

Antiviral activity of a novel mixture of natural antimicrobials, in vitro, and in a chicken infection model in vivo.

The aim of this study was to test in vitro the ability of a mixture of citrus extract, maltodextrin, sodium chloride, lactic acid and citric acid (AuraShield L) to inhibit the virulence of infectious bronchitis, Newcastle disease, avian influenza, porcine reproductive and respiratory syndrome (PRRS) and bovine coronavirus viruses. Secondly, in vivo, we have investigated its efficacy against infectious bronchitis using a broiler infection model. In vitro, these antimicrobials had expressed antiviral activity against all five viruses through all phases of the infection process of the host cells. In vivo, the antimicrobial mixture reduced the virus load in the tracheal and lung tissue and significantly reduced the clinical signs of infection and the mortality rate in the experimental group E2 receiving AuraShield L. All these effects were accompanied by a significant reduction in the levels of pro-inflammatory cytokines and an increase in IgA levels and short chain fatty acids (SCFAs) in both trachea and lungs. Our study demonstrated that mixtures of natural antimicrobials, such AuraShield L, can prevent in vitro viral infection of cell cultures. Secondly, in vivo, the efficiency of vaccination was improved by preventing secondary viral infections through a mechanism involving significant increases in SCFA production and increased IgA levels. As a consequence the clinical signs of secondary infections were significantly reduced resulting in recovered production performance and lower mortality rates in the experimental group E2.

Antiviral activity of green silver nanoparticles produced using aqueous buds extract of Syzygium aromaticum.

In the present study, we synthesized silver (Ag) nanoparticles using aqueous extracts of clove (Syzygium aromaticum) (SAE). This synthesis of green silver nanoparticles (AgNP) was a novel and effectual tool against the Newcastle Viral Disease (NDV). Syzygium aromaticum extract was used as reducing and stabilizing agent for synthesis of silver nanoparticles. AgNP were characterized using diversity of biophysical methods inclusive of Fourier transform infrared spectroscopy (FTIR), UV-VIS spectroscopy and Transmission electron microscopy (TEM) for morphology and size. Furthermore, XRD analysis confirmed the crystalline nature of the particles. In current investigations, the antiviral activity of clove buds silver nanoparticles was inspected in-vitro and in-ovo. Embryonated chicken eggs were used to perform the cytotoxicity assay of the clove extract silver nanoparticles (CESN). CESN showed in vitro antiviral activity against NDV in embryonated eggs.

In vitro and in vivo experimental trials to assess the modulatory influence of ß-caryophyllene on NDV replication and immunopathogenesis.

A trial was conducted to evaluate the antiviral activity and immunomodulatory effect of B-Caryophyllene (BCP) using NDV as a viral model. First, an in ovo experiment was conducted to estimate the antiviral mechanism of BCP. Next, an in vivo experiment was designed to confirm its antiviral efficacy as well as its immunomodulatory and growth promoting ability. According to the in ovo experiment, BCP possesses antiviral influence up to 61.7% when treated before or during NDV infection. Oral supplementation of chickens with two doses of BCP (200 and 400 µg/bird) resulted in a significant increase in the NDV HI-Ab responses and a significant increase in interferon-α signaling cytokines. These obvious immunomodulatory effects improved the bird clinical protection against virulent NDV challenge. To conclude, we introduced a new compound for the poultry industry sector that has antiviral and immunostimulant properties when supplemented orally before or during NDV infection.

Comparative Study of Protection against Newcastle Disease in Young Broilers Administered Natural Chicken Alpha Interferon via Oral and Intramuscular Routes.

Despite extensive vaccination approaches, Newcastle disease (ND) remains a permanent threat to the poultry industry worldwide. Besides vaccination, there is a burgeoning demand for new antivirals for use in interventions to control ND. One strategy is to strengthen the host innate immunity via host-derived innate immune proteins. Type I interferons define one of the first lines of innate immune defense against viral infections. Chicken interferon alpha (chIFN-α) is one of the potent cytokines that trigger antiviral responses. In the current study, we investigated the therapeutic effect of natural chIFN-α administered via oral and intramuscular (i.m.) routes against ND in broiler chickens. Our results showed that the level of protection against ND in response to chIFN-α therapy was dependent on the route and dose of IFN administration. A better therapeutic effect was observed in chickens treated with chIFN-α via the oral route than in those treated via the i.m. route. Regardless of the administration route, double-dose chIFN-α (2,000-U) treatments provided better protection than single-dose (1,000-U) treatments. However, complete protection against ND was achieved in birds treated with repeated doses of chIFN-α via the oral route. Histopathology of trachea, proventriculus, spleen, and liver showed a significant improvement in ND-induced degenerative changes in double-dose IFN-treatment groups compared to single-dose groups. Results of the hemagglutination test demonstrated a decrease in ND virus (NDV) titer in IFN-treated groups. Also, double doses of chIFN-α via oral route resulted in early recovery in weight gain. We propose that chIFN-α therapy via oral route could be an important therapeutic tool to control NDV infection in chicken.IMPORTANCE Newcastle disease (ND) is an economically important contagious disease of wild and domestic birds worldwide. The disease causes severe economic losses in terms of production due to high mortality and morbidity in nonvaccinated chickens. Despite extensive vaccination approaches, Newcastle disease (ND) remains a permanent threat to the poultry industry worldwide. In the current study, we used natural chicken IFN-α as an innate immune modulator to counteract ND in chickens. We report that chIFN-α is effective in protecting the chickens against ND and also prevents shedding of the virus, which can then prevent further spread of the disease. We propose that in addition to vaccination, chIFN-α therapy could be an effective option for controlling ND in areas of endemicity.

2ß-3,4-Unsaturated sialic acid derivatives: Synthesis optimization, and biological evaluation as Newcastle disease virus hemagglutinin-neuraminidase inhibitors.

The optimization of the synthetic protocol to obtain the 3,4-unsaturated sialic acid derivatives, through the fine-tuning of both the Ferrier glycosylation conditions and the subsequent hydrolysis work-up, is herein reported. The accomplishment of the desired ß-anomers and some selected α-ones, in pure form, led us to evaluate their specific inhibitory activity towards NDV-HN and human sialidase NEU3. Importantly, the resulting data allowed the identification, for the first time, of three active 3,4-unsaturated sialic acid analogs, showing IC50 values against NDV-HN in the micromolar range.

Therapeutic potential of Nitazoxanide against Newcastle disease virus: A possible modulation of host cytokines.

Newcastle disease (ND) is prevalent among the domesticated and the wild birds and is caused by the avian paramyxovirus serotype-I (APMV-I). It is commonly known to affect chicken, pheasant, ostrich, pigeon and waterfowl. Depending on the virulence, the velogenic NDV strains cause severe respiratory and nervous disorders with a high mortality rate. The live and killed vaccines are available for the prevention of infection in the market, but the drug for the treatment is not available. Nitazoxanide (NTZ), a member of thiazolides, is an antiparasitic drug. In the present study, the effect of NTZ on the NDV replication was explored. The experiments were conducted in chicken fibroblast cells (DF-1), PBMC, embryonated chicken eggs, and two-week old chickens. The inhibition of the NDV was observed upon post-treatment of NTZ at a concentration of ~12.5 µM. Cytokine profiling of the DF-1, PBMC, and chicken embryonic tissue treated with NTZ revealed significant upregulation in all the cytokines studied except for IL-1ß in DF-1 cells. It is plausible that NTZ is involved in causing immune-modulatory effects in poultry. NTZ treatment in two weeks old chicken showed significant reduction in NDV replication in trachea, and lungs, respectively, at 72 h post-infection. Encouraging results from the present study warrants repurposing NTZ as a drug for the treatment of viral infection in poultry. It will also pave the way towards understanding of similar effect against other animal pathogens.

Benefit of Dietary Supplementation with Bacillus subtilis BYS2 on Growth Performance, Immune Response, and Disease Resistance of Broilers.

A strain of Bacillus subtilis (B. subtilis) BYS2 was previously isolated from Mount Tai, which is located in Tai'an City in the Shandong Province of China. The strain was then stored in the Environmental Microbiology Laboratory at Shandong Agricultural University. To evaluate the effect of the bacterium preparation in broiler production, we fed the bacterium (106 CFU/g) to 1-day-old broilers and continued this feeding for 6 weeks to analyze its effect on growth and immune performance. We found that the average weight of the bacterium-fed group increased by 17.19% at weeks 5 compared to the control group (P < 0.05). The height of the villi in the duodenum and jejunum and the ratio of villi to crypt were significantly increased in the bacterium-fed group at weeks 5 (P < 0.05). Also, the IgG in the serum of broilers in the experimental group increased by 31.60% (P < 0.05) and IgM 30.52% (P < 0.05) compared with those in the control group. The expressions of the major pattern recognition receptors (PRRs), antiviral proteins, pro-inflammatory cytokines, and ß-defensins were significantly higher than those in the control group (P < 0.05). Meanwhile, the bursa immune organ indices of broilers in the experimental group were significantly higher than those in the control group (P < 0.05). Also, after 5 weeks of continuous feeding, when infected with Escherichia coli (E. coli) O1K1 and Newcastle disease virus (NDV) F48E8, the content of bacteria and virus in tissues and organs of the experimental group decreased significantly, and the survival rate of infected chickens increased by 31.1% and 17.7%, respectively (P < 0.05). These results show that the anti-infective B. subtilis BYS2 could, to some extent, replace antibiotics to promote growth, improve innate immunity, and enhance disease resistance in broilers.

Resiquimod inhibits Newcastle disease virus replication by modulating host cytokines: An understanding towards its possible therapeutics.

Newcastle disease virus (NDV) infects domestic and wild avian species with high mortality and morbidity worldwide. Although this disease is mainly controlled through NDV vaccines, alternative use of antiviral compounds is increasingly under study. Resiquimod (R-848), an imidazoquinoline compound is a potent synthetic agonist of Toll-like receptor 7 (TLR7). Until now reports regarding the adjuvant potential of resiquimod is well established against human viruses but has been less explored against avian viruses. In the present study, we have analysed the anti-NDV effect of resiquimod in chicken embryo fibroblast cells (DF-1) and embryonated chicken eggs. About 70% reduction in NDV replication was observed 48 h and 72 h post-resiquimod treatment in DF-1 cells. Furthermore, differential host genes expression was observed in resiquimod treated DF-1 cells, PBMCs, and tissue sample of chicken embryos at a different time point. Among all the analyzed genes, significant up-regulation of viperin, IFNα, IFNγ, IL-1ß, TNFα, IL18 were observed in its transcriptional level. Furthermore, resiquimod treatment showed NDV reduction in two weeks old chickens. About 61% and 38% reduction in NDV replication was observed 72 h post-infection in lungs and spleen, respectively. The study suggests the modulation of host innate immunity regulatory genes by resiquimod, which eventually modulates the NDV replication. The result of the study could be explored further to establish resiquimod as an alternative antiviral compound against NDV.

Identification of nucleoporin 93 (Nup93) that mediates antiviral innate immune responses.

RIG-I-like receptors (RLRs) are cytoplasmic sensors for viral RNA that elicit antiviral innate immune responses. RLR signaling culminates in the activation of the protein kinase TBK1, which mediates phosphorylation and nuclear translocation of IRF3 that regulates expression of type I interferon genes. Here, we found that Nucleoporin 93 (Nup93), components of nuclear pore complex (NPC), plays an important role in RLR-mediated antiviral responses. Nup93-deficient RAW264.7 macrophage cells exhibited decreased expression of Ifnb1 and Cxcl10 genes after treatment with a synthetic RLR agonist stimulation as well as Newcastle Disease Virus infection. Silencing Nup93 in murine primary macrophages and embryonic fibroblasts also resulted in reduced expression of these genes. IRF3 nuclear translocation during RLR signaling was impaired in Nup93-deficient RAW264.7 cells. Notably, the activation of TBK1 during RLR signaling was also decreased in Nup93-deficient cells. We found that Nup93 formed a complex with TBK1, and Nup93 overexpression enhanced TBK1-mediated IFNß promoter activation. Taken together, our findings suggest that Nup93 regulates antiviral innate immunity by enhancing TBK1 activity and IRF3 nuclear translocation.

Polyalthia longifolia leaves methanolic extract targets entry and budding of viruses-an in vitro experimental study against paramyxoviruses.

ETHNOPHARMACOLOGICAL RELEVANCE: Synthetic antiviral drugs have several limitations including high cost. Thus research on antiviral property of medicinal plants is continuously gaining importance. Polyalthia longifolia possesses several medicinal properties and has been used in traditional ayurvedic medicine for treatment of dermatological ailments as kushta, visarpa/herpes virus infection and also to treat pyrexia of unknown origin as mentioned in Visarpa Chikitsa. AIM OF THE STUDY: Keeping in view the cytotoxic, anti-cancer activity and antiviral efficacy of Polyalthia longifolia against herpes, present study was undertaken to evaluate the in vitro antiviral activity of methanolic extract of Polyalthia longifolia leaves, if any, and to unravel the possible target(s)/mechanism of action. MATERIAL AND METHODS: Antiviral activity of Polyalthia longifolia methanolic extract was studied using Vero cell lines against paramyxoviruses, namely-peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV). Cytotoxicity of the test extract was evaluated employing MTT assay. Virucidal activity, and viral-attachment, virus entry and release assays were determined in Vero cells using standard experimental protocols. The viral RNA in the virus-infected cells was quantified by qRT-PCR. RESULTS: At non-cytotoxic concentration, methanolic extract of Polyalthia longifolia leaves was found to inhibit the replication of PPRV and NDV at viral entry and budding level, whereas other steps of viral life cycle such as attachment and RNA synthesis remained unaffected. CONCLUSIONS: Polyalthia longifolia leaves extract possesses promising antiviral activity against paramyxoviruses and acts by inhibiting the entry and budding of viruses; and this plant extract evidently possesses excellent and promising potential for development of effective herbal antiviral drug.

Antiviral activities of extremophilic actinomycetes extracts from Kazakhstan's unique ecosystems against influenza viruses and paramyxoviruses.

BACKGROUND: Commercially available antiviral drugs, when used in the treatment of viral infections, do not always result in success. This is an urgent problem currently that needs to be addressed because several viruses including influenza and paramyxoviruses are acquiring multi-drug resistance. A potential solution for this emerging issue is to create new antiviral drugs from available compounds of natural products. It is known that the majority of drugs have been developed using compounds derived from actinomycetes, which are naturally occurring gram-positive bacteria. The purpose of this study was to investigate the antiviral properties of extremophilic actinomycetes extracts from strains that were isolated from extreme environments in Kazakhstan. METHODS: Five strains of extremophilic actinomycetes isolated from the unique ecosystems of Kazakhstan were extracted and tested for antiviral activity against influenza viruses (strains H7N1, H5N3, H1N1 and H3N2) and paramyxoviruses (Sendai Virus and Newcastle Disease Virus). The antiviral activity of these selected extracts was tested by checking their effect on hemagglutination and neuraminidase activities of the studied viruses. Additionally, actinomycetes extracts were compared with commercially available antiviral drugs and some plant preparations that have been shown to exhibit antiviral properties. RESULTS: The main findings show that extracts from strains K-192, K-340, K-362, K-522 and K525 showed antiviral activities when tested using influenza viruses, Sendai Virus, and Newcastle Disease Virus. These activities were comparable to those shown by Rimantadine and Tamiflu drugs, and "Virospan" and "Flavovir" plant preparations. CONCLUSIONS: We identified several extracts with antiviral activities against several strains of influenza viruses and paramyxoviruses. Our research findings can be applied towards characterization and development of new antiviral drugs from the active actinomycetes extracts.

Phosphorylated abacavir analogue (ABC-1) has ameliorative action against Newcastle disease virus induced pathogenesis in chicken.

Newcastle disease virus (NDV) causes huge economic loss to the poultry industry due to high mortality and morbidity. The present study aimed to assess the protective role of novel phosphorylated analogue ABC-1 in vivo in NDV-infected chickens through the inhibition of fusion protein. Both NDV-induced oxidative damage and protective role of novel phosphorylated ABC-1 were evaluated in vital organs such as the liver and lung of chickens. Enzyme linked immunosorbent assay (ELISA) results showed that protein oxidation and nitration levels were significantly raised in NDV-infected tissues compared to healthy controls, whereas these levels were reduced significantly (P < 0.05) in birds treated with phosphorylated compounds compared to the NDV-infected group alone. Additional investigation with double immunofluorescence showed that the large amount of immuno colocalization and Western blot analysis also confirmed this observation through its band pattern in NDV-infected birds compared to healthy birds, whereas these alterations were reduced in treatment with novel phosphorylated ABC-1. The expression of fusion glycoprotein was studied by immuno colocalization, PCR, and flow cytometry, and results demonstrated that the novel phosphorylated analogues reduced the expression of fusion glycoprotein. These results put forth that novel phosphorylated ABC-1 protects chickens from NDV-induced pathogenesis, protein oxidation/nitration, and exerts potent antiviral activity.

Host CARD11 Inhibits Newcastle Disease Virus Replication by Suppressing Viral Polymerase Activity in Neurons.

Host factors play multiple essential roles in the replication and pathogenesis of mammalian neurotropic viruses. However, the cellular proteins of the central nervous system (CNS) involved in avian neurotropic virus infection have not been completely elucidated. Here, we employed a gene microarray to identify caspase recruitment domain-containing protein 11 (CARD11), a lymphoma-associated scaffold protein presenting brain-specific upregulated expression in a virulent neurotropic Newcastle disease virus (NDV)-infected natural host. Chicken primary neuronal cells infected with NDV appeared slightly syncytial and died quickly. CARD11 overexpression inhibited viral replication and delayed cytopathic effects; conversely, depletion of CARD11 enhanced viral replication and cytopathic effects in chicken primary neuronal cells. The inhibition of viral replication by CARD11 could not be blocked with CARD11-Bcl10-MALT1 (CBM) signalosome and NF-κB signaling inhibitors. CARD11 was found to interact directly with the viral phosphoprotein (P) through its CC1 domain and the X domain of P; this X domain also mediated the interaction between P and the viral large polymerase protein (L). The CARD11 CC1 domain and L competitively bound to P via the X domain that hindered the P-L interaction of the viral ribonucleoprotein (RNP) complex, resulting in a reduction of viral polymerase activity in a minigenome assay and inhibition of viral replication. Animal experiments further revealed that CARD11 contributed to viral replication inhibition and neuropathology in infected chicken brains. Taken together, our findings identify CARD11 as a brain-specific antiviral factor of NDV infection in avian species.IMPORTANCE Newcastle disease virus (NDV) substantially impacts the poultry industry worldwide and causes viral encephalitis and neurological disorders leading to brain damage, paralysis, and death. The mechanism of interaction between this neurotropic virus and the avian central nervous system (CNS) is largely unknown. Here, we report that host protein CARD11 presented brain-specific upregulated expression that inhibited NDV replication, which was not due to CARD11-Bcl10-MALT1 (CBM) complex-triggered activation of its downstream signaling pathways. The inhibitory mechanism of viral replication is through the CARD11 CC1 domain, and the viral large polymerase protein (L) competitively interacts with the X domain of the viral phosphoprotein (P), which hampers the P-L interaction, suppressing the viral polymerase activity and viral replication. An in vivo study indicated that CARD11 alleviated neuropathological lesions and reduced viral replication in chicken brains. These results provide insight into the interaction between NDV infection and the host defense in the CNS and a potential antiviral target for viral neural diseases.

Inhibitory effect of grapefruit seed extract (GSE) on avian pathogens.

The inhibitory activities of grapefruit seed extract (GSE) on avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), Salmonella Infantis (SI) and Escherichia coli (EC) were evaluated. Original GSE contained 0.24% benzalkonium chloride (BZC), however, 0.0025% BZC solution could not inactivate bacteria. The activity of diluted GSE (×100, ×500 and ×1,000 with redistilled water) against selected viruses and bacteria was evaluated in this study. The GSE solutions were incubated with the pathogens over a period of time after which the remaining viruses were titrated and the bacterial colonies were counted. In the presence of organic material-5% fetal bovine serum (FBS), the test solutions were sprayed at 1 cm and 30 cm distances to test the efficacy of GSE in a spray form. Furthermore, the efficacy of GSE against bacteria on clothes was tested using non-woven cloth. GSE×100 reduced the viral titer of both AIV and NDV even in 5% FBS condition. IBDV showed high resistance to GSE. GSE×1,000 inactivated both SI and EC within 5 sec, even in the presence of 5% FBS. The disinfectant was able to maintain its efficacy in the spray form at 30 cm distance. GSE was also effective against SI and EC inoculated on fabric. GSE is a potential novel disinfectant against viruses and bacteria, effective even within a short contact time.

Efficacy of CpG-ODN Administration Routes on Humoral Responses against Newcastle disease in Broilers.

Un-methylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODN) has been considered as a powerful vaccine adjuvant and recognition of CpG-ODN by chicken leukocytes promotes their ability to fight against infections. In our study, efficacy of different routes of CpG-ODN application as an adjuvant on immune responses (antibody titer together with leukogram) following vaccination against Newcastle disease (ND) has been evaluated in broiler chickens (Ross-308). The results indicated that routes of CpG-ODN administration influence immune responses and comparison effectiveness of CpG-OND delivery routes showed that group vaccinated by eye-drop application had the highest antibody titer than that of the group injected intramuscularly (im) and the difference was significant (p = 0.04) on day 35 of age. Antibody titer of the group treated with Clone 30 plus CpG-ODN via eye-drop route was higher than that of the group vaccinated with clone 30 alone on days 28 and 35 of age and the difference was significant (p = 0.04). Co-administration of both vaccine and CpG improved outcome of leukogram of the chickens on days 21 to 42 of age and among the treated groups, WBC of the group received both vaccine and CpG by eye-drop route significantly (p &lt; 0.05) differed from that of the group vaccinated with clone 30 alone on days 28 and 35 but not on day 42 of age. Average final body weight of the control group did not significantly differ from those of the treated groups at end of the experiment. In conclusion, co-administration of ND vaccine plus CpG-ODN via eye-drop route improves immune responses.

Structural characterization, teratogenicity and in vitro avian antimicrobial activity of posterior salivary gland (PSG) toxin from cuttlefish, Sepia prashadi.

A low molecular weight posterior salivary gland (PSG) toxin was isolated and purified from the cuttlefish Sepia prashadi by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). The protein and neutral sugar content of the PSG toxin was determined to be 1.033 mg/g and 282 µg/g. Fourier Transform Infrared (FT-IR) spectroscopy revealed the presence of υ-OH, υ-CO and δ-NH functional groups. Circular Dichroism (CD) spectroscopy and K2D2 analysis quantified the presence of 38.39% α-helix and 9.25% ß-sheet and 52.36% of ß-turn. Matrix Assisted Laser Desorption/Ionization-Time-of Flight/Mass Spectrometry (MALDI-TOF/MS) and MASCOT analysis revealed the amino acid sequence of MEMQSKQQNSKAPANRKIFPWMKTSAVATASKRVEMASLLNLQERQIKIWFQNRMKQKSQQPQTR (1.92 kDa) homologous to homeobox protein H4 of pufferfish, T. rubripes. The PSG toxin showed differential stability with pH and induced premature hatching in Zebrafish eggs and dose dependant developmental malformations in embryos with a Maximum tolerated dose of 1.85 µM. The PSG toxin exhibited significant antibacterial activity with pronounced zone of inhibition against S. typhimurium (12.94 mm) and inhibited avian RBC binding of Newcastle Disease virus (NDV) at a titre value of 1/4. The present study strongly advocates the biomedical potential of the PSG toxin from S. prashadi and illustrates its promise as a potential avian antimicrobial agent of the future.

RETRACTED: Evaluation of imidazole and its derivative against Newcastle disease virus infection in chicken: A drug repurposing approach.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief and Author as, in addition to the Corrigendum already published in regards to this paper, many of the images were duplicated throughout the paper:­ Figure 1b: all four images of the 36hrs imidazole treatment group 10mM, 20mM, 30mM and 40mM, are the same as the images given in Figure 2C imidazole+virus group 10mM, 20mM, 30mM and 40mM. ­ Figure 2b: GFP image of 12hrs NDV+IMD is the same as the image given in Figure 2c 30mM GFP. ­ Figure 2h: all brightfield images for the first four panels are the same. ­ Figure 3b: plaques in the fourth and fifth panel are same. ­ Figure 3d: gross embryo image in the control (1st) is the same as NDV+ALB (4th panel) group. ­ Figure 4a: gross image 1 and 5 are the same.