A recombinant rabies virus expressing a phosphoprotein–eGFP fusion is rescued and applied to the rapid virus neutralization antibody assay

Tang, Hai-Bo; Lu, Zhuan-Ling; Wei, Xian-Kai; Zhong, Yi‐Zhi; Zhong, Tao; Pan, Yawen; Luo, Yang; Liao, Su-Huan; Minamoto, Nobuyuki; Luo, Ting

doi:10.1016/j.jviromet.2015.03.022

Cited by 10 publications

(5 citation statements)

References 17 publications

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“…In this study, 4-week-old mice were inoculated intracranially with a high titer of ERAGS-GFP, which was safe for 16 days and did not cause loss of body weight in mice. Recombinant RABV (rRV-eGFP), which expressed enhance GFP fused with RABV phosphoprotein and was constructed from RC-HL strain, showed similar safety in 4-week-old mice [ 13 ]. This suggests that ERAGS-GFP, which derives from a genetically modified RABV (ERAGS), is safer for use in a modified FAVN assay than CVS-11, which has neurotropic and virulent characteristics.…”

Section: Discussionmentioning

confidence: 99%

“…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%

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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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Section: Discussionmentioning

confidence: 99%

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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“…In contrast, lentiviruses expressing the luciferase enzyme require luciferase reporter kits and a luminometer to be quantified. 3) rLS1-1eGFP can be used for high throughput assays by measuring eGFP positive cells with a fluorescence reader directly in plates without any staining [ 18 , 20 , 21 ]. 4) Finally, rLS1-1eGFP vector can be used to express foreign F and HN in its natural context instead of using a surrogate system that may not reflect the same entry mechanism and therefore infectious efficacy.…”

Section: Discussionmentioning

confidence: 99%

Development of a novel Newcastle disease virus (NDV) neutralization test based on recombinant NDV expressing enhanced green fluorescent protein

Chumbe

Izquierdo-Lara

Calderón

et al. 2017

Virol J

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BackgroundNewcastle disease is one of the most important infectious diseases of poultry, caused by Newcastle disease virus (NDV). This virus is distributed worldwide and it can cause severe economic losses in the poultry industry due to recurring outbreaks in vaccinated and unvaccinated flocks. Protection against NDV in chickens has been associated with development of humoral response. Although hemagglutination inhibition (HI) assay and ELISA do not corroborate the presence of neutralizing antibodies (nAbs); they are used to measure protection and immune response against NDV.MethodsIn this study, we established a system to recover a recombinant NDV (rLS1) from a cloned cDNA, which is able to accept exogenous genes in desired positions. An enhanced green fluorescent protein (eGFP) gene was engineered in the first position of the NDV genome and we generated a recombinant NDV carrying eGFP. This NDV- eGFP reporter virus was used to develop an eGFP-based neutralization test (eGFP-NT), in which nAbs titers were expressed as the reciprocal of the highest dilution that expressed the eGFP.ResultsThe eGFP-NT gave conclusive results in 24 h without using any additional staining procedure. A total of 57 serum samples were assayed by conventional neutralization (NT) and eGFP-NT. Additionally, HI and a commercial ELISA kit were evaluated with the same set of samples. Although HI (R 2 = 0.816) and ELISA (R 2 = 0.791) showed substantial correlation with conventional NT, eGFP-NT showed higher correlation (R 2 = 0.994), indicating that eGFP-NT is more accurate method to quantify nAbs.ConclusionsOverall, the neutralization test developed here is a simple, rapid and reliable method for quantitation of NDV specific nAbs. It is suitable for vaccine studies and diagnostics.Electronic supplementary materialThe online version of this article (10.1186/s12985-017-0900-8) contains supplementary material, which is available to authorized users.

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“…The RFFIT test is rather time-consuming because cells have to be incubated for 24 h prior to detection of infected cells by direct immunofluorescence [ 30 ]. Recombinant RABV expressing GFP may facilitate detection of infected cells [ 34 , 35 , 36 ], but still is a hazardous pathogen. Pseudotyping of G-deficient, non-replicative RABV is also feasible but suffers from slower reporter gene expression [ 37 ].…”

Section: Discussionmentioning

confidence: 99%

Quantification of Lyssavirus-Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles

Moeschler¹,

Locher²,

Conzelmann

et al. 2016

Viruses

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Rabies is a highly fatal zoonotic disease which is primarily caused by rabies virus (RABV) although other members of the genus Lyssavirus can cause rabies as well. As yet, 14 serologically and genetically diverse lyssaviruses have been identified, mostly in bats. To assess the quality of rabies vaccines and immunoglobulin preparations, virus neutralization tests with live RABV are performed in accordance with enhanced biosafety standards. In the present work, a novel neutralization test is presented which takes advantage of a modified vesicular stomatitis virus (VSV) from which the glycoprotein G gene has been deleted and replaced by reporter genes. This single-cycle virus was trans-complemented with RABV envelope glycoprotein. Neutralization of this pseudotype virus with RABV reference serum or immune sera from vaccinated mice showed a strong correlation with the rapid fluorescent focus inhibition test (RFFIT). Importantly, pseudotype viruses containing the envelope glycoproteins of other lyssaviruses were neutralized by reference serum to a significantly lesser extent or were not neutralized at all. Taken together, a pseudotype virus system has been successfully developed which allows the safe, fast, and sensitive detection of neutralizing antibodies directed against different lyssaviruses.

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A recombinant rabies virus expressing a phosphoprotein–eGFP fusion is rescued and applied to the rapid virus neutralization antibody assay

Cited by 10 publications

References 17 publications

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

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

Development of a novel Newcastle disease virus (NDV) neutralization test based on recombinant NDV expressing enhanced green fluorescent protein

Quantification of Lyssavirus-Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles

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