Limits...
Newcastle disease virus fusion protein is the major contributor to protective immunity of genotype-matched vaccine.

Kim SH, Wanasen N, Paldurai A, Xiao S, Collins PL, Samal SK - PLoS ONE (2013)

Bottom Line: Although the current vaccines are substantially effective, they do not completely prevent infection, virus shedding and disease.The cleavage site of the Ban/010 F protein was mutated to the avirulent motif found in strain LaSota.However, only those chickens immunized with chimeric rLaSota expressing the F or F plus HN proteins of the Indonesian strain were efficiently protected against shedding of Ban/010 virus.

View Article: PubMed Central - PubMed

Affiliation: Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT
Virulent strains of Newcastle disease virus (NDV) can cause devastating disease in chickens worldwide. Although the current vaccines are substantially effective, they do not completely prevent infection, virus shedding and disease. To produce genotype-matched vaccines, a full-genome reverse genetics system has been used to generate a recombinant virus in which the F protein cleavage site has been changed to that of avirulent vaccine virus. In the other strategy, the vaccines have been generated by replacing the F and HN genes of a commercial vaccine strain with those from a genotype-matched virus. However, the protective efficacy of a chimeric virus vaccine has not been directly compared with that of a full-genome virus vaccine developed by reverse genetics. Therefore, in this study, we evaluated the protective efficacy of genotype VII matched chimeric vaccines by generating three recombinant viruses based on avirulent LaSota (genotype II) strain in which the open reading frames (ORFs) encoding the F and HN proteins were replaced, individually or together, with those of the circulating and highly virulent Indonesian NDV strain Ban/010. The cleavage site of the Ban/010 F protein was mutated to the avirulent motif found in strain LaSota. In vitro growth characteristics and a pathogenicity test indicated that all three chimeric viruses retained the highly attenuated phenotype of the parental viruses. Immunization of chickens with chimeric and full-length genome VII vaccines followed by challenge with virulent Ban/010 or Texas GB (genotype II) virus demonstrated protection against clinical disease and death. However, only those chickens immunized with chimeric rLaSota expressing the F or F plus HN proteins of the Indonesian strain were efficiently protected against shedding of Ban/010 virus. Our findings showed that genotype-matched vaccines can provide protection to chickens by efficiently preventing spread of virus, primarily due to the F protein.

Show MeSH

Related in: MedlinePlus

Cleavage of the F0 proteins of parental and chimeric viruses.(A) Proteolytic cleavage of the F0 proteins of parental and chimeric viruses in infected DF1 cells analyzed by Western blot. (B) The positions of the precursor protein F0, the cleavage product F1, and the HN protein are indicated. Lanes: 1. rLaSota, 2. rBan/AF, 3. rLaSota-Ban/AF F, 4. rLaSota-Ban/AF HN, and 5. rLaSota-Ban/AF F HN (B) The relative levels of the F0 and F1 proteins in the Western blot experiment in part C were measured by Bio-Rad Gel Image analysis, and the efficiency of cleavage was determined by dividing the amount of F1 by the amount of F1 plus F0. Each bar represents mean and standard error of the mean of triplicate samples.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3755997&req=5

pone-0074022-g003: Cleavage of the F0 proteins of parental and chimeric viruses.(A) Proteolytic cleavage of the F0 proteins of parental and chimeric viruses in infected DF1 cells analyzed by Western blot. (B) The positions of the precursor protein F0, the cleavage product F1, and the HN protein are indicated. Lanes: 1. rLaSota, 2. rBan/AF, 3. rLaSota-Ban/AF F, 4. rLaSota-Ban/AF HN, and 5. rLaSota-Ban/AF F HN (B) The relative levels of the F0 and F1 proteins in the Western blot experiment in part C were measured by Bio-Rad Gel Image analysis, and the efficiency of cleavage was determined by dividing the amount of F1 by the amount of F1 plus F0. Each bar represents mean and standard error of the mean of triplicate samples.

Mentions: Strain LaSota lacks a polybasic sequence or furin motif at the F protein cleavage site, and depends on extracellular protease for cleavage [19]. In our previous study, we modified the highly virulent Indonesian Ban/010 strain by changing its cleavage site sequence (RRQKR↓F) to that of LaSota (GRQGR↓L), resulting in the avirulent rBan/AF derivative. Whereas the Ban/010 parent induced extensive syncytia and plaque formation in cell culture in the absence of added protease, the rBan/AF derivative caused only single-cell infections without syncytia or plaques in the presence or absence of extracellular protease [17]. Therefore, in the present study, the ability of the chimeric viruses to form syncytia and plaques in cell culture was determined. DF1 cells were infected with the parental (rLaSota and rBan/AF) and the three chimeric viruses at an MOI of 0.01 in the presence or absence of 10% allantoic fluid as protease supplementation. The cells were visualized 48 hpi by photomicroscopy directly (Figure 2A) and following immunostaining with antiserum against the NDV N protein (Figure 2B). In parallel, the ability of the viruses to produce plaques was evaluated on DF1 cells under 0.8% methylcellulose overlay (not shown). In the presence of added protease, the rLaSota virus produced syncytia (Figure 2A and B) and plaques (not shown), whereas the rBan/AF virus produced neither. Among the three chimeric viruses, only the one containing F from the LaSota and HN from the rBan/AF strain (rLaSota-Ban/AF HN) produced syncytia and plaques, similar to those of rLaSota. In contrast, the other two chimeric viruses (rLaSota-Ban/AF F and rLaSota-Ban/AF F HN) produced neither syncytia (Figure 2A and B) nor plaques (not shown). Thus, the formation of syncytia and plaques in the presence of protease was associated with the presence of the LaSota F protein. Evaluation of the cleavage efficiency of the F proteins by Western blot analysis showed that the LaSota-derived F protein present in both rLaSota and rLaSota-Ban/AF HN was cleaved more efficiently than the rBan/AF-derived F protein present in the other viruses (Figure 3). These results suggest that the greater efficiency of cleavage of the LaSota-derived F protein was required for syncytium and plaque formation.


Newcastle disease virus fusion protein is the major contributor to protective immunity of genotype-matched vaccine.

Kim SH, Wanasen N, Paldurai A, Xiao S, Collins PL, Samal SK - PLoS ONE (2013)

Cleavage of the F0 proteins of parental and chimeric viruses.(A) Proteolytic cleavage of the F0 proteins of parental and chimeric viruses in infected DF1 cells analyzed by Western blot. (B) The positions of the precursor protein F0, the cleavage product F1, and the HN protein are indicated. Lanes: 1. rLaSota, 2. rBan/AF, 3. rLaSota-Ban/AF F, 4. rLaSota-Ban/AF HN, and 5. rLaSota-Ban/AF F HN (B) The relative levels of the F0 and F1 proteins in the Western blot experiment in part C were measured by Bio-Rad Gel Image analysis, and the efficiency of cleavage was determined by dividing the amount of F1 by the amount of F1 plus F0. Each bar represents mean and standard error of the mean of triplicate samples.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3755997&req=5

pone-0074022-g003: Cleavage of the F0 proteins of parental and chimeric viruses.(A) Proteolytic cleavage of the F0 proteins of parental and chimeric viruses in infected DF1 cells analyzed by Western blot. (B) The positions of the precursor protein F0, the cleavage product F1, and the HN protein are indicated. Lanes: 1. rLaSota, 2. rBan/AF, 3. rLaSota-Ban/AF F, 4. rLaSota-Ban/AF HN, and 5. rLaSota-Ban/AF F HN (B) The relative levels of the F0 and F1 proteins in the Western blot experiment in part C were measured by Bio-Rad Gel Image analysis, and the efficiency of cleavage was determined by dividing the amount of F1 by the amount of F1 plus F0. Each bar represents mean and standard error of the mean of triplicate samples.
Mentions: Strain LaSota lacks a polybasic sequence or furin motif at the F protein cleavage site, and depends on extracellular protease for cleavage [19]. In our previous study, we modified the highly virulent Indonesian Ban/010 strain by changing its cleavage site sequence (RRQKR↓F) to that of LaSota (GRQGR↓L), resulting in the avirulent rBan/AF derivative. Whereas the Ban/010 parent induced extensive syncytia and plaque formation in cell culture in the absence of added protease, the rBan/AF derivative caused only single-cell infections without syncytia or plaques in the presence or absence of extracellular protease [17]. Therefore, in the present study, the ability of the chimeric viruses to form syncytia and plaques in cell culture was determined. DF1 cells were infected with the parental (rLaSota and rBan/AF) and the three chimeric viruses at an MOI of 0.01 in the presence or absence of 10% allantoic fluid as protease supplementation. The cells were visualized 48 hpi by photomicroscopy directly (Figure 2A) and following immunostaining with antiserum against the NDV N protein (Figure 2B). In parallel, the ability of the viruses to produce plaques was evaluated on DF1 cells under 0.8% methylcellulose overlay (not shown). In the presence of added protease, the rLaSota virus produced syncytia (Figure 2A and B) and plaques (not shown), whereas the rBan/AF virus produced neither. Among the three chimeric viruses, only the one containing F from the LaSota and HN from the rBan/AF strain (rLaSota-Ban/AF HN) produced syncytia and plaques, similar to those of rLaSota. In contrast, the other two chimeric viruses (rLaSota-Ban/AF F and rLaSota-Ban/AF F HN) produced neither syncytia (Figure 2A and B) nor plaques (not shown). Thus, the formation of syncytia and plaques in the presence of protease was associated with the presence of the LaSota F protein. Evaluation of the cleavage efficiency of the F proteins by Western blot analysis showed that the LaSota-derived F protein present in both rLaSota and rLaSota-Ban/AF HN was cleaved more efficiently than the rBan/AF-derived F protein present in the other viruses (Figure 3). These results suggest that the greater efficiency of cleavage of the LaSota-derived F protein was required for syncytium and plaque formation.

Bottom Line: Although the current vaccines are substantially effective, they do not completely prevent infection, virus shedding and disease.The cleavage site of the Ban/010 F protein was mutated to the avirulent motif found in strain LaSota.However, only those chickens immunized with chimeric rLaSota expressing the F or F plus HN proteins of the Indonesian strain were efficiently protected against shedding of Ban/010 virus.

View Article: PubMed Central - PubMed

Affiliation: Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT
Virulent strains of Newcastle disease virus (NDV) can cause devastating disease in chickens worldwide. Although the current vaccines are substantially effective, they do not completely prevent infection, virus shedding and disease. To produce genotype-matched vaccines, a full-genome reverse genetics system has been used to generate a recombinant virus in which the F protein cleavage site has been changed to that of avirulent vaccine virus. In the other strategy, the vaccines have been generated by replacing the F and HN genes of a commercial vaccine strain with those from a genotype-matched virus. However, the protective efficacy of a chimeric virus vaccine has not been directly compared with that of a full-genome virus vaccine developed by reverse genetics. Therefore, in this study, we evaluated the protective efficacy of genotype VII matched chimeric vaccines by generating three recombinant viruses based on avirulent LaSota (genotype II) strain in which the open reading frames (ORFs) encoding the F and HN proteins were replaced, individually or together, with those of the circulating and highly virulent Indonesian NDV strain Ban/010. The cleavage site of the Ban/010 F protein was mutated to the avirulent motif found in strain LaSota. In vitro growth characteristics and a pathogenicity test indicated that all three chimeric viruses retained the highly attenuated phenotype of the parental viruses. Immunization of chickens with chimeric and full-length genome VII vaccines followed by challenge with virulent Ban/010 or Texas GB (genotype II) virus demonstrated protection against clinical disease and death. However, only those chickens immunized with chimeric rLaSota expressing the F or F plus HN proteins of the Indonesian strain were efficiently protected against shedding of Ban/010 virus. Our findings showed that genotype-matched vaccines can provide protection to chickens by efficiently preventing spread of virus, primarily due to the F protein.

Show MeSH
Related in: MedlinePlus