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Molecular and immunogenic characterization of BHK-21 cell line adapted CVS-11 strain of rabies virus and future prospect in vaccination strategy.

ABSTRACT: Development of a cost effective quality vaccine is a key issue in rabies control programme in developing countries. With this perspective, in the present study, challenge virus standard (CVS)-11 strain of rabies virus was adapted to grow in BHK-21 cells, characterized, compared with other viruses including global vaccine strains and field isolates from Indian subcontinent and China at molecular level. This cell adapted virus was evaluated for the production of cost effective veterinary vaccine. The maximum virus titre achieved was 10(7) fluorescent focus unit (FFU)/mL at 10th passage level. There was no nucleotide difference in the nucleoprotein (N) and glycoprotein (G) genes after adaptation in cell line. Phylogenetic analysis showed that adapted virus was grouped with global vaccine strains, closest being with other CVS strains but distinct from the Indian field isolates. Global vaccine strains including cell adapted CVS-11 virus have 83-87 % identity at nucleotide level of G gene with Indian field viruses. Growth kinetics of cell culture adapted virus showed that the optimum virus titer (around 10(7) FFU/mL) could be obtained at around 48 h post infection by co-cultivation method using 0.1 multiplicity of infection inoculums at 37 °C. These findings can be used for up scaling of vaccine production. The protective efficacy of test vaccine produced using 10(6.95) FFU/mL cell culture harvest showed 1.17 IU/mL relative potency by NIH test. Further, adapted virus was found to be suitable for use in rapid fluorescent focus inhibition test.

SUBMITTER: Patel AC

PROVIDER: S-EPMC4663707 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Molecular and immunogenic characterization of BHK-21 cell line adapted CVS-11 strain of rabies virus and future prospect in vaccination strategy.

Patel Arunkumar C AC Upmanyu Vikramaditya V Ramasamy Santhamani S Gupta Praveen Kumar PK Singh Rajendra R Singh Rabindra Prasad RP

Virusdisease 20151119 4

Development of a cost effective quality vaccine is a key issue in rabies control programme in developing countries. With this perspective, in the present study, challenge virus standard (CVS)-11 strain of rabies virus was adapted to grow in BHK-21 cells, characterized, compared with other viruses including global vaccine strains and field isolates from Indian subcontinent and China at molecular level. This cell adapted virus was evaluated for the production of cost effective veterinary vaccine. ...[more]

PMID: 26645040

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Project description:Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity, Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action.This work do not present or propose bufotenine as a drug for the treatment of rabies due to the hallucinogen and psychotropic effects of the molecule. However, continued studies in the elucidation of the antiviral mechanism of this molecule, may lead to the choice or development of a tryptamine analogue presenting potential clinical use.

Project description:BACKGROUND:Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms. METHODS:Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus - PV) strain was used for virological assays based on rapid fluorescent focus inhibition test. RESULTS:Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. < IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the virus's glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide. CONCLUSIONS:This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.

Dataset Information

Molecular and immunogenic characterization of BHK-21 cell line adapted CVS-11 strain of rabies virus and future prospect in vaccination strategy.

Publications

Molecular and immunogenic characterization of BHK-21 cell line adapted CVS-11 strain of rabies virus and future prospect in vaccination strategy.

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