First production of fluorescent anti‐ribonucleoproteins conjugate for diagnostic of rabies in Brazil

Caporale, Graciane Maria Medeiros; Silva, Andréa de Cássia Rodrigues da; Peixoto, Zélia Maria Pinheiro; Chaves, Luciana Botelho; Carrieri, Maria Luíza; Vassão, Ruth Camargo

doi:10.1002/jcla.20275

Cited by 24 publications

(9 citation statements)

References 17 publications

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“…Since FAT by using the anti-NP conjugate is much more sensitive than using conjugates specific to the total virion, we attempted to obtain this protein in its purified form, in order to produce the conjugate in the future ( 6 , 12 ). In this study the virus was obtained in a similar titer as previously described by a Brazilian study ( 10 ). The extracted rabies virus NP was confirmed by SDS-PAGE and Western blotting as the viral strain used in this project was the standard rabies virus strain (PV).…”

Section: Discussionmentioning

confidence: 99%

“…The extracted rabies virus NP was confirmed by SDS-PAGE and Western blotting as the viral strain used in this project was the standard rabies virus strain (PV). There was also some partial purification of NP, which resulted in a few minor bands on SDS-PAGE after purification with no probability of interference with the specific conjugate ( 10 , 12 ). Commercial conjugates are against the total virion or specific to the rabies NP (such as the conjugate from Bio-Rad, France), or they are prepared from monoclonal antibodies ( 3 ).…”

Section: Discussionmentioning

confidence: 99%

“…Then, the protein concentration was measured by a spectrophotometer at a wave length of 280 nm. In order to confirm NP presence, 5 µL of the sample of purified RNP was loaded on a 12.5% SDS-PAGE gel, with 5% stacking and stained with Coomassie blue (Merck, Germany) ( 10 , 15 ). We also performed a modified Western blot analysis using only the Bio-Rad conjugate (Bio-Rad, France) to stain a fixed blot of the extracted NP, as this conjugate is a specific antibody against the Rabies NP.…”

Section: Methodsmentioning

confidence: 99%

“…The world health organization (WHO) and the International Office of Epizootics (OIE) recommend the fluorescent antibody technique (FAT), which is done on specimens from central nervous system, skin biopsy and salivary glands as the preferable laboratory method and gold standard for diagnosis of rabies ( 6 , 9 , 10 ). The nucleoprotein (NP) with an approximate molecular weight of 50 KDa, consists of 450 amino acids, and it is the most preserved antigenic protein for different rabies strains ( 1 , 5 ).…”

Section: Introductionmentioning

confidence: 99%

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Cell Culture Extraction and Purification of Rabies Virus Nucleoprotein

Dastkhosh

Rahimi

Haghighat

et al. 2014

Jundishapur J Microbiol

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Background:Rabies is a major zoonotic viral disease and is detected using the World Health Organization standard diagnostic techniques. Rabies detection is preferably done using the fluorescent antibody technique (FAT) that provides reliable diagnosis with almost 100% accuracy for all variant strains, if a proper conjugate is used. Rabies virus nucleoprotein (NP) is the most important protein used in production of a specific diagnostic conjugate.Objectives:The aim of this study was to extract the cell-associated rabies virus NP from infected Baby Hamster Kidney cell clone (BSR) with rabies virus (Pasteur vaccine strain/PV) and purify for a future project to produce an anti-NP conjugate.Materials and Methods:Pasteur vaccine strain (PV) as the standard rabies vaccine strain with a focus-forming dose (FFD) of 105 was inoculated in to the BSR cell culture at a concentration of 106 cells per milliliter. Infected cells were harvested 72 hours after infection and the rabies NP was extracted from these cells by low-speed centrifugation and purification by ultracentrifugation in cesium chloride (CsCl) gradient. For analysis, the purified NP was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Results:The volume of the lysate was 15 mL and it became 2.5 mL after purification, with a concentration of 3.25 mg/mL. The corresponding band to the cell lysate protein on the SDS-PAGE had a molecular weight of 50 KDa, similar to the molecular weight of NP in rabies virus.Conclusions:The rabies virus NP could be extracted and purified in an appropriate amount from infected cell culture. The results of SDS-PAGE analysis showed that the intact rabies virus NP had been purified properly and thus could be used for further steps to produce the specific diagnostic rabies conjugate.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cell Culture Extraction and Purification of Rabies Virus Nucleoprotein

Dastkhosh

Rahimi

Haghighat

et al. 2014

Jundishapur J Microbiol

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“…[ 24 ]. The staining was carried out by adding 40 μL of an optimal dilution of the antirabies fluorescent conjugate [ 25 ]. After one hour of incubation, the microtiter plates were washed by immersion in PBS and then in distilled water.…”

Section: Methodsmentioning

confidence: 99%

Bufotenine is able to block rabies virus infection in BHK-21 cells

Vigerelli

Sciani

Jared

et al. 2014

J Venom Anim Toxins incl Trop Dis

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BackgroundRabies 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.MethodsBufotenine 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.ResultsIn 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1678-9199-20-45) contains supplementary material, which is available to authorized users.

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In vitro effects of bufotenine against RNA and DNA viruses

et al. 2021

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Bufotenine, an alkaloid that can be found in plant extracts and skin secretions of amphibians, is reported to have potential antiviral activity. The present study evaluated the antiviral activity of bufotenine against different genetic lineages of rabies virus (RABV, a single-stranded, negative-sense RNA virus), canine coronavirus (CCoV, a positive-sense RNA virus) and two double-stranded DNA viruses (two strains of herpes simplex virus type 1/HSV-1 [KOS and the acyclovir-resistant HSV-1 strain 29R] and canine adenovirus 2, CAV-2). The maximal non-toxic bufotenine concentrations in Vero and BHK-21 cells were determined by MTT assays. The antiviral activity of bufotenine against each virus was assessed by examination of reductions in infectious virus titres and plaque assays. All experiments were performed with and without bufotenine, and the results were compared. Bufotenine demonstrated significant RABV inhibitory activity. No antiviral action was observed against CCoV, CAV-2 or HSV-1. These findings indicate that the antiviral activity of bufotenine is somewhat linked to the particular infectious dose used and the genetic lineage of the virus, although the mechanisms of its effects remain undetermined.

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First production of fluorescent anti‐ribonucleoproteins conjugate for diagnostic of rabies in Brazil

Cited by 24 publications

References 17 publications

Cell Culture Extraction and Purification of Rabies Virus Nucleoprotein

Cell Culture Extraction and Purification of Rabies Virus Nucleoprotein

Bufotenine is able to block rabies virus infection in BHK-21 cells

In vitro effects of bufotenine against RNA and DNA viruses

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