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Protective antibody response of Balb/c mice to Bali rabies virus isolate propagated in BHK-21 cells.

ABSTRACT: The protective antibody response of Balb/c mice to Bali rabies virus (RABV) in BHK-21 cells was studied. The virus was isolated from a rabid dog and was adapted to replicate in BHK-21 cell culture for seven passages. The BHK-21-adapted Bali RABV (BHK-Bali RABV) was inactivated with binary ethylenimine and 24 mice were immunized twice at 21-days intervals with the inactivated virus and Rabisin® vaccine. Virus replication was detected using indirect immunofluorescence, immunocytochemistry, and western blotting assays. Enzyme-linked immunosorbent assay examination 2 weeks after the first immunization revealed RABV antibody titers that were mostly below the minimum protective level (<0.5 equivalent unit, EU). Antibody titers increased sharply after the second immunization. Antibody titers in serum of mice induced by inactivated BHK-Bali RABV one week after the second immunization were slightly lower (0.8-3.8 EU) than those induced by Rabisin vaccine (0.9-6.3 EU). RABV antibody titers were stable for at least 6 weeks after the second immunization. Both Rabisin vaccine and inactivated BHK-Bali RABV induced neutralizing antibodies with neutralization titers (50% protective dose per ml) of 29.84 for 0.1 ml Rabisin, 211.41 for 0.2 ml Rabisin, 27.41 for 0.1 ml BHK-Bali RABV, and 28.25 for 0.2 ml BHK-Bali RABV. Thus, inactivated BHK-Bali RABV induces a protective immune response in Balb/c mice, but at lower levels compared to induction by Rabisin vaccine.

SUBMITTER: Astawa INM

PROVIDER: S-EPMC6207530 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Protective antibody response of Balb/c mice to Bali rabies virus isolate propagated in BHK-21 cells.

Astawa I Nyoman Mantik INM Agustini Ni Luh Putu NLP Masa Tenaya I Wayan IW Aryawiguna I Putu Gede Widnyana IPGW

The Journal of veterinary medical science 20180911 10

The protective antibody response of Balb/c mice to Bali rabies virus (RABV) in BHK-21 cells was studied. The virus was isolated from a rabid dog and was adapted to replicate in BHK-21 cell culture for seven passages. The BHK-21-adapted Bali RABV (BHK-Bali RABV) was inactivated with binary ethylenimine and 24 mice were immunized twice at 21-days intervals with the inactivated virus and Rabisin® vaccine. Virus replication was detected using indirect immunofluorescence, immunocytochemistry, and wes ...[more]

PMID: 30210066

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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:BackgroundRabies 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.MethodsSkin 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.ResultsHerein, 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.ConclusionsThis novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.

Project description:BackgroundPreviously thought to be rabies free, Bali experienced an outbreak of animal and human rabies cases in November 2008. We describe the epidemiological and clinical data of human rabies cases occurring in the first two years of the outbreak.MethodsWe analysed the patient records of all rabies cases from the Sanglah General Hospital in Denpasar, and district hospitals in Buleleng and Tabanan. A conventional reverse transcriptase polymerase chain reaction was developed to detect the rabies virus genome in saliva, corneal swabs, and ante- and post-mortem cerebrospinal fluid (CSF).ResultsThere were 104 human rabies cases in Bali during November 2008-November 2010. Patients' mean age was 36.6 years (range 3-84 years; SD 20.7), most were male (56.7%), and originated from rural districts. Almost all (92%) cases had a history of dog bite. Only 5.8% had their wounds treated and received an anti-rabies vaccine (ARV) after the bite incident. No patients received rabies immunoglobulin (RIG). The estimated time from dog bite to the onset of signs and symptoms was 110.4 days (range 12-720 days; SD 118.2). The mean length of medical care until death was 21.8 hours (range 1-220 hours; SD 32.6). Less than 50% of patients had prodromal symptoms. The most frequent prodromal symptom was pain or paraesthesia at the bite site (37.6%). The two most common central nervous system infection signs were agitation (89.2%) and confusion (83.3%). Signs of autonomic nervous system dysfunction included hydrophobia (93.1%), hypersalivation (88.2%), and dyspnea (74.4%). On admission, 22 of 102 patients (21.6%) showed paralytic manifestations, while the rest (78.4%) showed furious rabies manifestations. The case-fatality rate was 100%. The rabies virus genome was detected in 50 of 101 patients (49.5%) with the highest detection rate from post-mortem CSF samples.ConclusionsRabies is a major public health problem in Bali. Human fatalities occur because of a lack of knowledge regarding rabies risk, the poor management of dog bites, and the limited availability of RIG. Increasing public awareness of dog bite management, increasing the availability of ARV and RIG, and implementing an island-wide dog vaccination campaign will help prevent human rabies cases.

Dataset Information

Protective antibody response of Balb/c mice to Bali rabies virus isolate propagated in BHK-21 cells.

Publications

Protective antibody response of Balb/c mice to Bali rabies virus isolate propagated in BHK-21 cells.

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