Development and validation of a real-time RT-PCR assay for the quantification of rabies virus as quality control of inactivated rabies vaccines

Moreira, Beatriz Lourenç Correia; Pereira, Luciane Aparecida; Gimenez, Ana Paula Lappas; Inagaki, Hiroki; Raboni, Sônia Mara

doi:10.1016/j.jviromet.2019.04.025

Cited by 9 publications

(4 citation statements)

References 16 publications

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“…This is the first study employing TaqMan probe-based real-time PCR for the quantification of KFDV in vaccines. However, in vitro methods have been used to quantify virus load in other non-cytopathic virus vaccines; Lourenc et al [30] used TaqMan quantitative PCR to quantify rabies virus in cell culture vaccine harvests. Chabaud-riou et al [31] have used G protein-based ELISA for the estimation of rabies viral antigen load in vaccines and for evaluating potency tests of the rabies vaccine.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Safety and Potency of Kyasanur Forest Disease (KFD) Vaccine Inactivated with Different Concentrations of Formalin and Comparative Evaluation of In Vitro and In Vivo Methods of Virus Titration in KFD Vaccine

Srikanth,

Marinaik,

Gomes

et al. 2023

Biomedicines

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We evaluated the safety and potency of the Kyasanur Forest disease (KFD) vaccine inactivated with different formalin concentrations in mice, since the side effects due to higher formalin concentrations have been a major reason for vaccine refusal. Furthermore, with an objective to reduce the use of mice in vaccine testing, we performed quantification of the KFD virus by real-time PCR and compared it with in vivo titration in mice. The KFD vaccine prepared in chicken embryo fibroblast cells was inactivated with 0.04%, 0.06%, and 0.08% concentrations of formalin. The vaccine inactivated with 0.04% and 0.06% formalin failed the safety test, whereas the KFD vaccine inactivated with 0.08% formalin was safe and potent with a log protective index of 5678 in mice. This reduced formalin content may induce no/lesser side-effects of pain/swelling which may increase the vaccine acceptance. The real-time PCR on individual KFD vaccine harvests interpreted that when the CT value of each harvest is <20, the vaccine will have sufficient viral particles to pass the potency test. Comparison of the real-time PCR on tenfold dilutions of the pooled harvests with in vivo mice inoculation test revealed that the 1MLD50 of the vaccine lies in the tenfold dilution that yields CT values between 31 and 34.

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Section: Discussionmentioning

confidence: 99%

Evaluation of Safety and Potency of Kyasanur Forest Disease (KFD) Vaccine Inactivated with Different Concentrations of Formalin and Comparative Evaluation of In Vitro and In Vivo Methods of Virus Titration in KFD Vaccine

Srikanth,

Marinaik,

Gomes

et al. 2023

Biomedicines

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“…The recombinant viruses were continuously passaged in suspended BHK-21 cells, and the supernatants and cells were collected every five passages for subsequent virus titration and genetic stability detection. The viral titers of recombinant viruses were detected by a previously described method ( Jin et al, 2021 ) and calculated according to the Reed-Muench method ( Muench and Reed, 1938 ; Ahmad et al, 2017 ; Lourenc Correia Moreira et al, 2019 ; Liu et al, 2020 ). The identified recombinant viruses were purified by a previously described method ( Jin et al, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

An inactivated recombinant rabies virus chimerically expressed RBD induces humoral and cellular immunity against SARS-CoV-2 and RABV

Zhang¹,

Jin²,

Yan³

et al. 2023

Virologica Sinica

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“…The CFP_8011 primer set was described previously by Gudra et al [ 10 ]. The β-actin primer set was modified from Moreira et al [ 25 ]. The primers and probes used in this study are shown in Table 1 .…”

Section: Methodsmentioning

confidence: 99%

Recombinant Virus Quantification Using Single-Cell Droplet Digital PCR: A Method for Infectious Titer Quantification

Korotkaja

Zajakina

2023

Viruses

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The quantification of viruses is necessary for both research and clinical applications. The methods available for RNA virus quantification possess several drawbacks, including sensitivity to inhibitors and the necessity of a standard curve generation. The main purpose of this study was to develop and validate a method for the quantification of recombinant, replication-deficient Semliki Forest virus (SFV) vectors using droplet digital PCR (ddPCR). This technique demonstrated stability and reproducibility using various sets of primers that targeted inserted transgenes, as well as the nsP1 and nsP4 genes of the SFV genome. Furthermore, the genome titers in the mixture of two types of replication-deficient recombinant virus particles were successfully measured after optimizing the annealing/extension temperature and virus:virus ratios. To measure the infectious units, we developed a single-cell ddPCR, adding the whole infected cells to the droplet PCR mixture. Cell distribution in the droplets was investigated, and β-actin primers were used to normalize the quantification. As a result, the number of infected cells and the virus infectious units were quantified. Potentially, the proposed single-cell ddPCR approach could be used to quantify infected cells for clinical applications.

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Development and validation of a real-time RT-PCR assay for the quantification of rabies virus as quality control of inactivated rabies vaccines

Cited by 9 publications

References 16 publications

Evaluation of Safety and Potency of Kyasanur Forest Disease (KFD) Vaccine Inactivated with Different Concentrations of Formalin and Comparative Evaluation of In Vitro and In Vivo Methods of Virus Titration in KFD Vaccine

Evaluation of Safety and Potency of Kyasanur Forest Disease (KFD) Vaccine Inactivated with Different Concentrations of Formalin and Comparative Evaluation of In Vitro and In Vivo Methods of Virus Titration in KFD Vaccine

An inactivated recombinant rabies virus chimerically expressed RBD induces humoral and cellular immunity against SARS-CoV-2 and RABV

Recombinant Virus Quantification Using Single-Cell Droplet Digital PCR: A Method for Infectious Titer Quantification

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