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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 replication of VSV pseudotypes on helper cell lines. (a) The helper cell line expressing the G protein of Street-Alabama-Dufferin B19 (SAD B19) was treated for 8 h with doxycycline (DOX; filled symbols) or were left untreated (open symbols). Following infection of the cells with VSV*∆G(FLuc) (multiplicity of infection (m.o.i.) of 0.05), cell culture supernatants were collected at the indicated times and virus titers determined on Vero cells taking advantage of green fluorescent protein (GFP) reporter expression; (b) Pseudotype virus yield on induced and non-induced helper cells. Helper cell lines expressing the G proteins either of VSV, SAD B19, challenge virus standard 11 (CVS-11), EBLV-1, EBLV-2, or MOKV were treated for 8 h with DOX or were left untreated. The cells were infected VSV*∆G(FLuc) (dark bars) and VSV*∆G(sNLuc) (grey bars) using an m.o.i. of 0.05. Cell culture supernatants were collected at 36 h post infection (p.i.) and titrated on Vero cells; (c) Pseudotype viruses were produced on T-Rex™-CHO helper cells expressing the G protein of SAD B19. Vero cells were infected with either VSV*∆G(FLuc) (black bars) or VSV*∆G(sNLuc) (grey bars) using the indicated m.o.i. At 6 h p.i., luciferase activity was measured in cell lysates and cell culture supernatant, respectively; (d) Time kinetics of luciferase reporter expression. Vero cells were infected with SAD B19 G protein-pseudotyped VSV*∆G(FLuc) (black plots) or VSV*∆G(sNLuc) (grey plots) using an m.o.i. of 0.01. Luciferase activity was measured at the indicated times in cell lysates and cell culture supernatants, respectively; (e) Vero cells were grown in 96-well microtiter plates and infected with VSV*∆G(sNLuc) using m.o.i. ranging from 0.0001 to 0.001. The number of GFP-expressing cells was enumerated in 95 wells and plotted against the Nano luciferase activity that was secreted into the cell culture supernatant of the respective wells. The dashed line indicates the luminescence cutoff value above which the cells of a well were regarded as infected. f.f.u.: focus-forming units; RLU: relative light units. * p < 0.05.
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viruses-08-00254-f002: Conditional replication of VSV pseudotypes on helper cell lines. (a) The helper cell line expressing the G protein of Street-Alabama-Dufferin B19 (SAD B19) was treated for 8 h with doxycycline (DOX; filled symbols) or were left untreated (open symbols). Following infection of the cells with VSV*∆G(FLuc) (multiplicity of infection (m.o.i.) of 0.05), cell culture supernatants were collected at the indicated times and virus titers determined on Vero cells taking advantage of green fluorescent protein (GFP) reporter expression; (b) Pseudotype virus yield on induced and non-induced helper cells. Helper cell lines expressing the G proteins either of VSV, SAD B19, challenge virus standard 11 (CVS-11), EBLV-1, EBLV-2, or MOKV were treated for 8 h with DOX or were left untreated. The cells were infected VSV*∆G(FLuc) (dark bars) and VSV*∆G(sNLuc) (grey bars) using an m.o.i. of 0.05. Cell culture supernatants were collected at 36 h post infection (p.i.) and titrated on Vero cells; (c) Pseudotype viruses were produced on T-Rex™-CHO helper cells expressing the G protein of SAD B19. Vero cells were infected with either VSV*∆G(FLuc) (black bars) or VSV*∆G(sNLuc) (grey bars) using the indicated m.o.i. At 6 h p.i., luciferase activity was measured in cell lysates and cell culture supernatant, respectively; (d) Time kinetics of luciferase reporter expression. Vero cells were infected with SAD B19 G protein-pseudotyped VSV*∆G(FLuc) (black plots) or VSV*∆G(sNLuc) (grey plots) using an m.o.i. of 0.01. Luciferase activity was measured at the indicated times in cell lysates and cell culture supernatants, respectively; (e) Vero cells were grown in 96-well microtiter plates and infected with VSV*∆G(sNLuc) using m.o.i. ranging from 0.0001 to 0.001. The number of GFP-expressing cells was enumerated in 95 wells and plotted against the Nano luciferase activity that was secreted into the cell culture supernatant of the respective wells. The dashed line indicates the luminescence cutoff value above which the cells of a well were regarded as infected. f.f.u.: focus-forming units; RLU: relative light units. * p < 0.05.

Mentions: For pseudotyping of VSV with rhabdoviral glycoproteins, either recombinant VSV*∆G(FLuc) expressing the two reporter genes GFP and FLuc [28] or VSV*∆G(sNLuc) expressing GFP and secreted sNLuc was used. In order to assess the efficacy of pseudotype particle production, the T-Rex™-CHO(SAD-G) helper cell line was infected with VSV*∆G(FLuc) using a multiplicity of infection (m.o.i.) of 0.05 infectious particles per cell. Aliquots of cell culture supernatants were collected at 12, 24, 36, 48, and 60 h post infection (p.i.) and infectious titers determined on Vero cells. Doxycycline-treated T-Rex™-CHO(SAD-G) cells produced maximum titers of about 108 fluorescent focus-forming units/mL (f.f.u./mL) at 48 h p.i., whereas only low amounts of virus (<102 f.f.u./mL) were released in the absence of doxycycline (Figure 2a). To determine the yield of pseudotype virus on different helper cell lines, the cells were infected with VSV*∆G(FLuc) or VSV*∆G(sNLuc) using an m.o.i. of 0.05. Cell culture supernatants were collected at 36 h p.i. and infectious virus titers determined on Vero cells. As expected, the highest virus yield (>108 f.f.u./mL) was obtained with T-Rex™-CHO(VSV-G) cells (Figure 2b). The pseudotype virus titers achieved on helper cell lines expressing lyssavirus G proteins ranged from 107 to 108 f.f.u./mL. However, T-Rex™-CHO(CVS11-G) cells produced significantly lower pseudotype virus titers (about 106 f.f.u./mL). This might be attributed to the pronounced neurotropism of CVS-11 and possibly lower expression levels of appropriate receptors in CHO cells. On all helper cell lines, infectious titers of VSV*∆G(FLuc) and VSV*∆G(sNLuc) did not differ (Figure 2b). Of note, significant levels of infectious pseudotype virus were only produced in the presence of doxycycline, indicating that the viruses were not able to propagate in the absence of G protein. Accordingly, non-helper cells (e.g., Vero cells) did not release any progeny virus following infection with any of the pseudotype viruses (data not shown).


Quantification of Lyssavirus -Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles
Conditional replication of VSV pseudotypes on helper cell lines. (a) The helper cell line expressing the G protein of Street-Alabama-Dufferin B19 (SAD B19) was treated for 8 h with doxycycline (DOX; filled symbols) or were left untreated (open symbols). Following infection of the cells with VSV*∆G(FLuc) (multiplicity of infection (m.o.i.) of 0.05), cell culture supernatants were collected at the indicated times and virus titers determined on Vero cells taking advantage of green fluorescent protein (GFP) reporter expression; (b) Pseudotype virus yield on induced and non-induced helper cells. Helper cell lines expressing the G proteins either of VSV, SAD B19, challenge virus standard 11 (CVS-11), EBLV-1, EBLV-2, or MOKV were treated for 8 h with DOX or were left untreated. The cells were infected VSV*∆G(FLuc) (dark bars) and VSV*∆G(sNLuc) (grey bars) using an m.o.i. of 0.05. Cell culture supernatants were collected at 36 h post infection (p.i.) and titrated on Vero cells; (c) Pseudotype viruses were produced on T-Rex™-CHO helper cells expressing the G protein of SAD B19. Vero cells were infected with either VSV*∆G(FLuc) (black bars) or VSV*∆G(sNLuc) (grey bars) using the indicated m.o.i. At 6 h p.i., luciferase activity was measured in cell lysates and cell culture supernatant, respectively; (d) Time kinetics of luciferase reporter expression. Vero cells were infected with SAD B19 G protein-pseudotyped VSV*∆G(FLuc) (black plots) or VSV*∆G(sNLuc) (grey plots) using an m.o.i. of 0.01. Luciferase activity was measured at the indicated times in cell lysates and cell culture supernatants, respectively; (e) Vero cells were grown in 96-well microtiter plates and infected with VSV*∆G(sNLuc) using m.o.i. ranging from 0.0001 to 0.001. The number of GFP-expressing cells was enumerated in 95 wells and plotted against the Nano luciferase activity that was secreted into the cell culture supernatant of the respective wells. The dashed line indicates the luminescence cutoff value above which the cells of a well were regarded as infected. f.f.u.: focus-forming units; RLU: relative light units. * p < 0.05.
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Related In: Results  -  Collection

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viruses-08-00254-f002: Conditional replication of VSV pseudotypes on helper cell lines. (a) The helper cell line expressing the G protein of Street-Alabama-Dufferin B19 (SAD B19) was treated for 8 h with doxycycline (DOX; filled symbols) or were left untreated (open symbols). Following infection of the cells with VSV*∆G(FLuc) (multiplicity of infection (m.o.i.) of 0.05), cell culture supernatants were collected at the indicated times and virus titers determined on Vero cells taking advantage of green fluorescent protein (GFP) reporter expression; (b) Pseudotype virus yield on induced and non-induced helper cells. Helper cell lines expressing the G proteins either of VSV, SAD B19, challenge virus standard 11 (CVS-11), EBLV-1, EBLV-2, or MOKV were treated for 8 h with DOX or were left untreated. The cells were infected VSV*∆G(FLuc) (dark bars) and VSV*∆G(sNLuc) (grey bars) using an m.o.i. of 0.05. Cell culture supernatants were collected at 36 h post infection (p.i.) and titrated on Vero cells; (c) Pseudotype viruses were produced on T-Rex™-CHO helper cells expressing the G protein of SAD B19. Vero cells were infected with either VSV*∆G(FLuc) (black bars) or VSV*∆G(sNLuc) (grey bars) using the indicated m.o.i. At 6 h p.i., luciferase activity was measured in cell lysates and cell culture supernatant, respectively; (d) Time kinetics of luciferase reporter expression. Vero cells were infected with SAD B19 G protein-pseudotyped VSV*∆G(FLuc) (black plots) or VSV*∆G(sNLuc) (grey plots) using an m.o.i. of 0.01. Luciferase activity was measured at the indicated times in cell lysates and cell culture supernatants, respectively; (e) Vero cells were grown in 96-well microtiter plates and infected with VSV*∆G(sNLuc) using m.o.i. ranging from 0.0001 to 0.001. The number of GFP-expressing cells was enumerated in 95 wells and plotted against the Nano luciferase activity that was secreted into the cell culture supernatant of the respective wells. The dashed line indicates the luminescence cutoff value above which the cells of a well were regarded as infected. f.f.u.: focus-forming units; RLU: relative light units. * p < 0.05.
Mentions: For pseudotyping of VSV with rhabdoviral glycoproteins, either recombinant VSV*∆G(FLuc) expressing the two reporter genes GFP and FLuc [28] or VSV*∆G(sNLuc) expressing GFP and secreted sNLuc was used. In order to assess the efficacy of pseudotype particle production, the T-Rex™-CHO(SAD-G) helper cell line was infected with VSV*∆G(FLuc) using a multiplicity of infection (m.o.i.) of 0.05 infectious particles per cell. Aliquots of cell culture supernatants were collected at 12, 24, 36, 48, and 60 h post infection (p.i.) and infectious titers determined on Vero cells. Doxycycline-treated T-Rex™-CHO(SAD-G) cells produced maximum titers of about 108 fluorescent focus-forming units/mL (f.f.u./mL) at 48 h p.i., whereas only low amounts of virus (<102 f.f.u./mL) were released in the absence of doxycycline (Figure 2a). To determine the yield of pseudotype virus on different helper cell lines, the cells were infected with VSV*∆G(FLuc) or VSV*∆G(sNLuc) using an m.o.i. of 0.05. Cell culture supernatants were collected at 36 h p.i. and infectious virus titers determined on Vero cells. As expected, the highest virus yield (>108 f.f.u./mL) was obtained with T-Rex™-CHO(VSV-G) cells (Figure 2b). The pseudotype virus titers achieved on helper cell lines expressing lyssavirus G proteins ranged from 107 to 108 f.f.u./mL. However, T-Rex™-CHO(CVS11-G) cells produced significantly lower pseudotype virus titers (about 106 f.f.u./mL). This might be attributed to the pronounced neurotropism of CVS-11 and possibly lower expression levels of appropriate receptors in CHO cells. On all helper cell lines, infectious titers of VSV*∆G(FLuc) and VSV*∆G(sNLuc) did not differ (Figure 2b). Of note, significant levels of infectious pseudotype virus were only produced in the presence of doxycycline, indicating that the viruses were not able to propagate in the absence of G protein. Accordingly, non-helper cells (e.g., Vero cells) did not release any progeny virus following infection with any of the pseudotype viruses (data not shown).

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