A new recombinant rabies virus expressing a green fluorescent protein: A novel and fast approach to quantify virus neutralizing antibodies

Qin, Shan; Volokhov, Dmitriy V.; Rodionova, Elvira; Wirblich, Christoph; Schnell, Matthias J.; Chizhikov, Vladimir E.; Dabrazhynetskaya, Alena

doi:10.1016/j.biologicals.2019.03.002

Cited by 14 publications

(4 citation statements)

References 26 publications

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“…С позиции повышения мер биобезопасности актуальными представляются модификации указанных методов, предполагающие использование непатогенных штаммов ВБ или близкородственных вирусов, например вируса везикулярного стоматита [20]. Другим аспектом совершенствования методов FAVN и RFFIT является применение генно-модифицированных штаммов ВБ, продуцирующих флуоресцирующий белок [21]. Такой подход позволяет исключить этап окрашивания клеточной культуры и сократить время исследования при условии сохранения надежности анализа in vitro.…”

Section: исследование вируснейтрализующей активности: Rffit и Favn-тестыunclassified

<i>In vitro</i> methods for rabies virus detection, and evaluation of their use in the production of rabies immunoglobulin

Гаврилова

Генералов

Абрамова

et al. 2021

Biological Products. Prevention, Diagnosis, Treatment

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Current highly sensitive methods for rabies virus and rabies antibodies detection in biological material can be used not only for diagnosis and experimental research, but also for the production of antirabies medicines used for postexposure prophylaxis. The aim of the study was to analyse existing methods for rabies virus and rabies antibodies detection and to assess the potential for using these methods at the control stages during production of heterologous antirabies immunoglobulin obtained from equine serum. The search for cutting-edge highly sensitive in vitro control methods that could compete with the biological method, which is the main method used in antirabies immunoglobulin control, is an important prerequisite for improvement of the production technology and the quality of antirabies medicines. The study demonstrated that the following test methods can be used in the production of antirabies immunoglobulin: fluorescent antibody technique, enzyme-linked immunosorbent assay, cell culture methods, atomic force microscopy, and flow cytometry. These methods could be used alone or as an alternative to the biological method in white mice. These methods were chosen because of their high sensitivity, specificity, rapid and easy implementation, cost-effectiveness, and automatic recording of test results.

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Section: исследование вируснейтрализующей активности: Rffit и Favn-тестыunclassified

<i>In vitro</i> methods for rabies virus detection, and evaluation of their use in the production of rabies immunoglobulin

Гаврилова

Генералов

Абрамова

et al. 2021

Biological Products. Prevention, Diagnosis, Treatment

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“…GFP is used to detect expression of proteins and for virus rescue [ 8 ]. Several recombinant RABVs expressing GFP have been generated by reverse genetics using the HEP-Flury, CVS-11, RC-HL, CVS-N2c, and Evelyn-Rokitnicki-Abelseth (ERA) strains [ 9 10 11 12 13 14 ]. Although these recombinant RABV had similar growth characteristics to the parent virus, the level of GFP expression varied with the genomic location of the GFP gene.…”

Section: Introductionmentioning

confidence: 99%

“…Although these recombinant RABV had similar growth characteristics to the parent virus, the level of GFP expression varied with the genomic location of the GFP gene. Recombinant RABVs expressing GFP were detected in infected cells from 24 to 48 post-inoculation [ 12 ]. It is known that the pathogenicity of RABV is related to the 333th amino acid of the RABV glycoprotein (G).…”

Section: Introductionmentioning

confidence: 99%

Generation of a recombinant rabies virus expressing green fluorescent protein for a virus neutralization antibody assay

et al. 2021

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Background Fluorescent antibody virus neutralization (FAVN) test is a standard assay for quantifying rabies virus-neutralizing antibody (VNA) in serum. However, a safer rabies virus (RABV) should be used in the FAVN assay. There is a need for a new method that is economical and time-saving by eliminating the immunostaining step. Objectives We aimed to improve the traditional FAVN method by rescuing and characterizing a new recombinant RABV expressing green fluorescent protein (GFP). Methods A new recombinant RABV expressing GFP designated as ERAGS-GFP was rescued using a reverse genetic system. Immuno-fluorescence assay, peroxidase-linked assay, electron microscopy and reverse transcription polymerase chain reaction were performed to confirm the recombinant ERAGS-GFP virus as a RABV expressing the GFP gene. The safety of ERAGS-GFP was evaluated in 4-week-old mice. The rabies VNA titers were measured and compared with conventional FAVN and FAVN-GFP tests using VERO cells. Results The virus propagated in VERO cells was confirmed as RABV expressing GFP. The ERAGS-GFP showed the highest titer (10 8.0 TCID 50 /mL) in VERO cells at 5 days post-inoculation, and GFP expression persisted until passage 30. The body weight of 4-week-old mice inoculated intracranially with ERAGS-GFP continued to increase and the survival rate was 100%. In 62 dog sera, the FAVN-GFP result was significantly correlated with that of conventional FAVN ( r = 0.95). Conclusions We constructed ERAGS-GFP, which could replace the challenge virus standard-11 strain used in FAVN test.

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“…The CVS-11 virus is used as a challenge virus in the RFFIT test to measure rabies neutralizing antibodies in vaccinated individuals' sera [17][18][19]. This test is the only gold standard serological assay recommended by WHO 2021 Vol.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Multiplicity of Infection (MOI) and Harvesting Time on the Production of CVS-11 Strain of Rabies Virus in BSR Cell Line

Rahpeyma

Bashar

2021

JoMMID

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Rabies is a zoonotic fatal viral disease caused by the rabies virus of the genus Lyssavirus, and the family Rhabdoviridae. Challenge virus standard (CVS-11) strain of rabies virus is a key element in rabies reference laboratories, as many gold-standard tests depend on a suitable titer of this strain for interpretation of results. The present study investigated the optimal CVS-11 production in BSR cells (a clone of BHK-21). Methods: We analyzed the kinetic growth of BSR cells in a T-flask and inoculated BSR cells with different MOI of CVS-11 strain of rabies virus, and harvested the produced virus at different time points. Results: Our data showed that BSR cells had a doubling time of around 24-30 h, and at least 95% of cells kept their viability three days after culture. The virus reached the highest titer when the cells were infected at an MOI of 0.1 in DMEM medium, equal to 1.5 × 107 fluorescent focus units (FFU)/ml. Time-course analysis of CVS-11 titer showed that the highest titer was achieved around 72 h post-infection. All tests were performed in triplicate. Conclusion: Since producing the virus in mammalian cell culture is an expensive and complicated method, optimizing the virus production process may be an excellent strategy to lower the cost, save the laboratory resources and maximize productivity.

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A new recombinant rabies virus expressing a green fluorescent protein: A novel and fast approach to quantify virus neutralizing antibodies

Cited by 14 publications

References 26 publications

<i>In vitro</i> methods for rabies virus detection, and evaluation of their use in the production of rabies immunoglobulin

<i>In vitro</i> methods for rabies virus detection, and evaluation of their use in the production of rabies immunoglobulin

Generation of a recombinant rabies virus expressing green fluorescent protein for a virus neutralization antibody assay

Evaluation of Multiplicity of Infection (MOI) and Harvesting Time on the Production of CVS-11 Strain of Rabies Virus in BSR Cell Line

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