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

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Performance of the PVN compared to the modified RFFIT. The PVN test makes use of propagation-incompetent pseudotype particles that express two reporter proteins, GFP and luciferase. The modified RFFIT makes use of live RABV and requires high biosafety standards. The different read-outs and time requirements of the assays are indicated. TCID50: median tissue culture infective dose.
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viruses-08-00254-f005: Performance of the PVN compared to the modified RFFIT. The PVN test makes use of propagation-incompetent pseudotype particles that express two reporter proteins, GFP and luciferase. The modified RFFIT makes use of live RABV and requires high biosafety standards. The different read-outs and time requirements of the assays are indicated. TCID50: median tissue culture infective dose.

Mentions: In the present work, virus-neutralizing antibodies were detected taking advantage of propagation-incompetent pseudotype virus particles that comply with biosafety level 1 or 2 (depending on the country you are working in). The rapid and strong expression of VSV-encoded reporter genes (GFP, luciferase) facilitated the detection of virus-infected cells and allowed the assay to be performed within 8 h (Figure 5). In particular, the luciferase reporter turned out to be highly sensitive and allowed detection of even a single infected cell. The secreted Nano luciferase turned out to be superior over the firefly luciferase as it did not require prior lysis of the cells and produced stronger signals. Luminescence was recorded using a plate reader, which allowed the analysis of several samples in short time. The definition of a luciferase cutoff value enabled us to discriminate between positive and negative wells and to calculate ND50 values that were similar to those obtained with the GFP read-out (see Figure 3b). As an alternative read-out, the half maximal inhibitory serum/antibody concentration (IC50) leading to 50% reduction of luciferase reporter activity may be calculated. This would allow detection of even lower levels of neutralizing antibodies, however, the data cannot be directly compared with standard ND50 values. Compared to the conventional RFFIT and FAVN tests, which require a lot of counting of cells/foci with the risk of individual mistakes by the experimenter, the PVN test with the automated luciferase detection has the advantage of better reproducibility and provides the opportunity of better standardization.


Quantification of Lyssavirus -Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles
Performance of the PVN compared to the modified RFFIT. The PVN test makes use of propagation-incompetent pseudotype particles that express two reporter proteins, GFP and luciferase. The modified RFFIT makes use of live RABV and requires high biosafety standards. The different read-outs and time requirements of the assays are indicated. TCID50: median tissue culture infective dose.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035968&req=5

viruses-08-00254-f005: Performance of the PVN compared to the modified RFFIT. The PVN test makes use of propagation-incompetent pseudotype particles that express two reporter proteins, GFP and luciferase. The modified RFFIT makes use of live RABV and requires high biosafety standards. The different read-outs and time requirements of the assays are indicated. TCID50: median tissue culture infective dose.
Mentions: In the present work, virus-neutralizing antibodies were detected taking advantage of propagation-incompetent pseudotype virus particles that comply with biosafety level 1 or 2 (depending on the country you are working in). The rapid and strong expression of VSV-encoded reporter genes (GFP, luciferase) facilitated the detection of virus-infected cells and allowed the assay to be performed within 8 h (Figure 5). In particular, the luciferase reporter turned out to be highly sensitive and allowed detection of even a single infected cell. The secreted Nano luciferase turned out to be superior over the firefly luciferase as it did not require prior lysis of the cells and produced stronger signals. Luminescence was recorded using a plate reader, which allowed the analysis of several samples in short time. The definition of a luciferase cutoff value enabled us to discriminate between positive and negative wells and to calculate ND50 values that were similar to those obtained with the GFP read-out (see Figure 3b). As an alternative read-out, the half maximal inhibitory serum/antibody concentration (IC50) leading to 50% reduction of luciferase reporter activity may be calculated. This would allow detection of even lower levels of neutralizing antibodies, however, the data cannot be directly compared with standard ND50 values. Compared to the conventional RFFIT and FAVN tests, which require a lot of counting of cells/foci with the risk of individual mistakes by the experimenter, the PVN test with the automated luciferase detection has the advantage of better reproducibility and provides the opportunity of better standardization.

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.