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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.


Related in: MedlinePlus

Conditional expression of rhabdoviral G proteins by transgenic helper cell lines. T-Rex™-Chinese Hamster Ovary (CHO) cells were stably transfected with recombinant pcDNA™ 4/TO plasmid encoding the indicated envelope G protein gene, selected with antibiotics, and cloned by limiting dilution. The cell clones were treated for 8 h with doxycycline (DOX) and inoculated with vesicular stomatitis virus (VSV)*ΔG. Clones that supported replication were selected and analyzed for expression of G protein: (a) Cell surface expression of the envelope G proteins was detected by indirect immunofluorescence using mAb I1 (VSV G protein), mAb 16DB4 (for detection of the G proteins of rabies virus (RABV), European bat lyssavirus type 1 (EBLV-1), and European bat lyssavirus type 2 (EBLV-2)), and rabbit anti-MOKV (Mokola virus) G protein serum as primary reagents; (b) Analysis of G protein expression by Western blot. Selected cell clones were treated with DOX for 8 h or left untreated, labeled with a membrane-impermeable biotinylation reagent, and lysed with NP-40 buffer. Solubilized cellular proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and blotted onto a nitrocellulose membrane. After incubating the membrane with a streptavidin-IRDye800 conjugate, the biotinylated cell surface proteins were detected with an Odyssey infrared scanner.
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viruses-08-00254-f001: Conditional expression of rhabdoviral G proteins by transgenic helper cell lines. T-Rex™-Chinese Hamster Ovary (CHO) cells were stably transfected with recombinant pcDNA™ 4/TO plasmid encoding the indicated envelope G protein gene, selected with antibiotics, and cloned by limiting dilution. The cell clones were treated for 8 h with doxycycline (DOX) and inoculated with vesicular stomatitis virus (VSV)*ΔG. Clones that supported replication were selected and analyzed for expression of G protein: (a) Cell surface expression of the envelope G proteins was detected by indirect immunofluorescence using mAb I1 (VSV G protein), mAb 16DB4 (for detection of the G proteins of rabies virus (RABV), European bat lyssavirus type 1 (EBLV-1), and European bat lyssavirus type 2 (EBLV-2)), and rabbit anti-MOKV (Mokola virus) G protein serum as primary reagents; (b) Analysis of G protein expression by Western blot. Selected cell clones were treated with DOX for 8 h or left untreated, labeled with a membrane-impermeable biotinylation reagent, and lysed with NP-40 buffer. Solubilized cellular proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and blotted onto a nitrocellulose membrane. After incubating the membrane with a streptavidin-IRDye800 conjugate, the biotinylated cell surface proteins were detected with an Odyssey infrared scanner.

Mentions: It has been previously shown that VSV lacking the glycoprotein G (VSVΔG) can be efficiently trans-complemented with the VSV G protein using a transgenic cell line [22]. This cell line was engineered to express the VSV G protein in a conditional manner in order to accommodate the cytotoxic properties of the protein [32]. Here, a similar approach was used for pseudotyping of VSVΔG with lyssavirus envelope glycoproteins. T-Rex™-CHO cells were transfected with the pcDNA™ 4/TO/Myc-His plasmid vector encoding the G protein of either VSV (serotype Indiana), rabies challenge virus standard 11 (CSV-11), rabies virus vaccine strain Street-Alabama-Dufferin B19 (SAD-B19), EBLV-1, EBLV-2, or MOKV (strain Ethiopia-16). The cells were selected with the antibiotics zeocin and blasticidin and subsequently cloned by limiting dilution. Following induction of transgene expression by doxycycline, cell clones were selected for their ability to support propagation of a recombinant G-deleted VSV expressing GFP (VSV*ΔG). The expression of the viral envelope glycoproteins by the selected cell clones was investigated by indirect immunofluorescence (Figure 1a). All G proteins could be detected only if the cells had been treated before with doxycycline. The antibodies reacted with intact, non-permeabilized cells indicating that the G proteins were expressed at the cell surface. Western blot analysis revealed that the G proteins had the expected molecular mass (Figure 1b). Using this approach, very little expression of G proteins was observed in non-induced cells.


Quantification of Lyssavirus -Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles
Conditional expression of rhabdoviral G proteins by transgenic helper cell lines. T-Rex™-Chinese Hamster Ovary (CHO) cells were stably transfected with recombinant pcDNA™ 4/TO plasmid encoding the indicated envelope G protein gene, selected with antibiotics, and cloned by limiting dilution. The cell clones were treated for 8 h with doxycycline (DOX) and inoculated with vesicular stomatitis virus (VSV)*ΔG. Clones that supported replication were selected and analyzed for expression of G protein: (a) Cell surface expression of the envelope G proteins was detected by indirect immunofluorescence using mAb I1 (VSV G protein), mAb 16DB4 (for detection of the G proteins of rabies virus (RABV), European bat lyssavirus type 1 (EBLV-1), and European bat lyssavirus type 2 (EBLV-2)), and rabbit anti-MOKV (Mokola virus) G protein serum as primary reagents; (b) Analysis of G protein expression by Western blot. Selected cell clones were treated with DOX for 8 h or left untreated, labeled with a membrane-impermeable biotinylation reagent, and lysed with NP-40 buffer. Solubilized cellular proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and blotted onto a nitrocellulose membrane. After incubating the membrane with a streptavidin-IRDye800 conjugate, the biotinylated cell surface proteins were detected with an Odyssey infrared scanner.
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Related In: Results  -  Collection

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viruses-08-00254-f001: Conditional expression of rhabdoviral G proteins by transgenic helper cell lines. T-Rex™-Chinese Hamster Ovary (CHO) cells were stably transfected with recombinant pcDNA™ 4/TO plasmid encoding the indicated envelope G protein gene, selected with antibiotics, and cloned by limiting dilution. The cell clones were treated for 8 h with doxycycline (DOX) and inoculated with vesicular stomatitis virus (VSV)*ΔG. Clones that supported replication were selected and analyzed for expression of G protein: (a) Cell surface expression of the envelope G proteins was detected by indirect immunofluorescence using mAb I1 (VSV G protein), mAb 16DB4 (for detection of the G proteins of rabies virus (RABV), European bat lyssavirus type 1 (EBLV-1), and European bat lyssavirus type 2 (EBLV-2)), and rabbit anti-MOKV (Mokola virus) G protein serum as primary reagents; (b) Analysis of G protein expression by Western blot. Selected cell clones were treated with DOX for 8 h or left untreated, labeled with a membrane-impermeable biotinylation reagent, and lysed with NP-40 buffer. Solubilized cellular proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and blotted onto a nitrocellulose membrane. After incubating the membrane with a streptavidin-IRDye800 conjugate, the biotinylated cell surface proteins were detected with an Odyssey infrared scanner.
Mentions: It has been previously shown that VSV lacking the glycoprotein G (VSVΔG) can be efficiently trans-complemented with the VSV G protein using a transgenic cell line [22]. This cell line was engineered to express the VSV G protein in a conditional manner in order to accommodate the cytotoxic properties of the protein [32]. Here, a similar approach was used for pseudotyping of VSVΔG with lyssavirus envelope glycoproteins. T-Rex™-CHO cells were transfected with the pcDNA™ 4/TO/Myc-His plasmid vector encoding the G protein of either VSV (serotype Indiana), rabies challenge virus standard 11 (CSV-11), rabies virus vaccine strain Street-Alabama-Dufferin B19 (SAD-B19), EBLV-1, EBLV-2, or MOKV (strain Ethiopia-16). The cells were selected with the antibiotics zeocin and blasticidin and subsequently cloned by limiting dilution. Following induction of transgene expression by doxycycline, cell clones were selected for their ability to support propagation of a recombinant G-deleted VSV expressing GFP (VSV*ΔG). The expression of the viral envelope glycoproteins by the selected cell clones was investigated by indirect immunofluorescence (Figure 1a). All G proteins could be detected only if the cells had been treated before with doxycycline. The antibodies reacted with intact, non-permeabilized cells indicating that the G proteins were expressed at the cell surface. Western blot analysis revealed that the G proteins had the expected molecular mass (Figure 1b). Using this approach, very little expression of G proteins was observed in non-induced cells.

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.


Related in: MedlinePlus