Growth of Rabies Virus in Cell Culture

Rabies virus from the submandibular salivary gland of a naturally infected fox was adapted to growth in BHK-21 cells. The pathogenicity of the original isolate and the cell culture adapted virus were compared by the intramuscular and oral routes in mice and foxes. Animals surviving exposure were tested for serum rabies antibodies (immunogenic efficiency) and for their ability to survive a second challenge with rabies virus (protective efficiency). In mice, ratios between lethal and protective doses of the two strains via the oral or muscular route were similar. In foxes, however, pathogenic efficiency was modified after cell culture adaptation, although good immunogenic and protective efficiency was maintained. Inoculation via the oral route resulted in lethal infection with the wild-type strain and survival (with some subsequent immunity) with the cell-adapted strain. Via the intramuscular route, foxes given high doses of cell culture adapted virus survived whereas several foxes given lower doses died and virus could not be reisolated. It is concluded that safety testing of modified strains should be done with different dilutions of virus in the target species. "Autosterilizing" infections may result in erroneous diagnosis.

Download Full-text

Calculating rabies virus neutralizing antibodies titres by flow cytometry

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652002000300007 ◽

2002 ◽

Vol 44 (3) ◽

pp. 151-154 ◽

Cited By ~ 14

Author(s):

Juliano BORDIGNON ◽

Fabiano COMIN ◽

Sílvia Córdoba P. FERREIRA ◽

Graciane M. M. CAPORALE ◽

José Hermênio Cavalcante LIMA FILHO ◽

...

Keyword(s):

Cell Culture ◽

Flow Cytometry ◽

Rabies Virus ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Standard Test ◽

Serum Samples ◽

Reference Serum ◽

Infection Inhibition

The determination of the rabies neutralizing antibody (VNA) response after immunization against rabies is an acceptable index of the efficacy of a vaccine and a successful treatment. Several tests have been developed in attempt to improve the assessment of VNA, from mice inoculation to cell-culture fluorescence inhibition tests. All of them, however, present special difficulties in terms of reading or accuracy. The present study describes a neutralization test performed in cell-culture appraised by flow cytometry (FC). Serial dilutions of the serum samples were mixed in vitro with rabies virus before the addition of BHK-21 cells. After 24h-incubation, cells were released by trypsin treatment, fixed and permeabilized with a p-formaldehyde solution and stained with a rabies virus nucleocapsid protein-specific antibody conjugate. The percentage of virus infection inhibition caused by specific antibodies present in the serum were evaluated in a Beckton & Dickinson FACSCalibur® flow cytometer. A correlation curve between the IU/ml content and the percentage of infective inhibition was built with a reference serum and the VNA titers of serum samples were obtained by extrapolation. Titers obtained by FC and standard test showed an effective pairing results (p < 0.01), with a correlation coefficient (r) = 0.7. These results permit to envisage the FC as a suitable technique to evaluate VNA in sera from immunized animals and likely in human serum samples. Nevertheless, new studies comparing FC to gold-standard techniques are required for determining the FC values of Sensibility and Specificity .

Download Full-text

Development of the scheme of obtaining antibodies to the ribonucleoprotein of attenuated rabies virus

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2019-5-3-8 ◽

2019 ◽

pp. 3-8 ◽

Cited By ~ 1

Author(s):

Yu. K. Gavrilova ◽

S. V. Generalov ◽

M. N. Kireev ◽

N. A. Sharapova ◽

E. G. Abramova ◽

...

Keyword(s):

Cell Culture ◽

Rabies Virus ◽

Antibody Titer ◽

Colloidal Gold ◽

Target Component ◽

High Antibody ◽

Experimental Animals ◽

Colloidal Gold Nanoparticles ◽

High Antibody Titer ◽

Dot Immunoassay

Aim. Isolation of ribonucleoprotein (RNP) of an attenuated rabies virus, develop schemes for immunizing animals with RNP-based preparations and determine the most effective scheme that allows obtaining serum with a high antibody titer to the RNP.Materials and methods. We used the transplantable cell line Vero, the strain rabies virus «Moscow 3253 Vero», adapted for reproduction on Vero, rabbits of the chinchilla breed. In order to obtain serums containing antibodies to the RNP of the rabies virus, experimental schemes have been proposed for immunizing animals with RNP, including with adjuvants: polyoxidonium and colloidal gold. The dynamics of the accumulation of antibodies to the RNP of the rabies virus in the blood serum of experimental animals was studied by dot-immunoassay.Results. The target component (RNP of the rabies virus) was isolated directly from the cytoplasm of the Vero cell culture infected with the rabies virus according to the modified M. Dastkhosh (2014) method, lyophilized and used in the development of preparations for immunizing experimental animals. In the study of the dynamics of the formation of antibodies to RNP of the rabies virus by the method of dot-immunoassay, the effectiveness of an adjuvant is established — colloidal gold nanoparticles ranging in size from 15 to 17 nm, the use of which makes it possible to increase the antibody titer by 2 times.Conclusion. The results obtained are of interest for further research related to the design of diagnostic products and the development of methodological techniques using such preparations.

Download Full-text

Optimization of Specifications for Scaled-Up Fixed Rabies Virus Cultivation (“Moscow 3253” Strain) in Vero Cell Culture

Problems of Particularly Dangerous Infections ◽

10.21055/0370-1069-2014-2-101-103 ◽

2014 ◽

pp. 101-103

Author(s):

S. V. Generalov ◽

E. G. Abramova ◽

Zh. V. Matveeva ◽

I. M. Zhulidov ◽

L. V. Savitskaya ◽

...

Keyword(s):

Cell Culture ◽

Vero Cell ◽

Rabies Virus ◽

Vero Cell Culture

Download Full-text

Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner

Viruses ◽

10.3390/v13101989 ◽

2021 ◽

Vol 13 (10) ◽

pp. 1989

Author(s):

Sabine Nitschel ◽

Luca M. Zaeck ◽

Madlin Potratz ◽

Tobias Nolden ◽

Verena te Kamp ◽

...

Keyword(s):

Cell Culture ◽

Cell Lines ◽

Rabies Virus ◽

Point Mutations ◽

Inverse Correlation ◽

Virus Release ◽

Independent Manner ◽

Adaptive Mutations ◽

Field Virus

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.

Download Full-text