Progress 10/01/99 to 09/30/04
Outputs
Field isolates of fowlpox virus (FPV) contain reticuloendotheliosis (REV) provirus in their genome while the vaccine strains have long terminal repeats (LTR) of REV. REV has been associated with tumor formation and immunosuppression. Field strains of FPV vary in pathogenicity. With a view to improve the current FPV vaccine, two recombinant vaccines were created. Studies with two genetically modified FPV vaccines (a) with removal of all REV sequences and (b) containing REV envelope gene have provided encouraging results. Fowlpox virus persists in the poultry environment for a long time. In this regard a gene encoding for DNA repair enzyme photolyase was identified in the genome of fowlpox virus. Presence of this enzyme seems to enhance stability of virus in the environment. Since replication of photolyase-deficient fowlpox virus is somewhat impaired in poultry, this mutant virus can be useful as an eco-friendly vaccine. Two genes which encode for A-type inclusion body
proteins identified in the genome of fowlpox virus are non-essential for viral replication. Perhaps the encoded proteins shield the virus from environmental damage. A putative hemaglutinin (HA) gene identified in the genome of all fowlpox viruses appears non-essential for replication. These non-essential loci can be used in the generation of recombinant FPV vaccines expressing antigens from multiple pathogens. In this regard, we have also identified several homologous fowlpox virus promoters. Homologous fowlpox virus promoters can be genetically manipulated to create recombinant vaccines in which expression of foreign gene(s) is maximally optimized to create effective poultry vaccines.
Impacts
Genetically modified fowlpox virus vaccine lacking REV sequences eliminate chances of future integration of these REV sequences in FPV genome. Persistence and stability of FPV in the environment can be minimized by deletion of photolyase and A-type inclusion genes. Effective recombinant vaccines for poultry can be developed by using homologous FPV promoters and non-essential loci in the FPV genome.
Publications
- Singh, P., Kim, T.-J. and Tripathy, D.N. 2000. Re-emerging fowlpox: Evaluation of isolates from vaccinated flocks. Avian Pathology 29: 449-455.
- Kim, T.J. and Tripathy, D.N. 2001. Reticuloendotheliosis virus integration in the fowlpoxvirus genome: Not a recent event. Avian Dis. 45: 663-669.
- Srinivasan, V., Schnitzlein, W.M. and Tripathy, D.N. 2001. Fowlpox virus encodes a novel DNA repair enzyme, CPD-photolyase, that restores infectivity of UV light-damaged virus. Journal of Virology 75:1681-1688.
- Shivprasad, H.L., Kim, T.-J., Woolcock, P.R. and Tripathy, D.N. 2002. Genetic and antigenic characterization of a poxvirus isolate from ostriches. Avian Dis. 46: 429-436.
- Wang, X., Schnitzlein, W.M, Tripathy, D.N., Girshick, T. and Khan, M.I. 2002. Construction and immunogenicity studies of recombinant fowlpox virus containing the S1gene of Massachusetts 41 strain of IBV. Avian Dis. 46 831-838.
- Singh, P., Kim, T.-J. and Tripathy, D.N. 2003. Identification and characterization of fowlpox virus strains using monoclonal antibodies. J. Vet. Diag.Ivest.15:50-54.
- Singh, P., Schnitzlein, W.M. and Tripathy, D.N. 2003. Reticuloendotheliosis virus sequences within the genomes of field strains of fowlpox virus display variability. J. Virology 77:5855-5862.
- Singh, P. and Tripathy, D.N. 2003. Fowlpox virus infection causes a lymphoproliferative response in chickens. Viral Immunology 16: 223-227.
- Srinivasan, V., Schnitzlein, W.M. and Tripathy, D.N. 2003. A consideration of previously uncharacterized fowlpox virus unidirectional and bi-directional promoters for inclusion in homologous recombinant vaccines. Avian Dis. 47: 286-295.
- Tripathy, D.N. 2003. The impact of vaccines and future of genetically modified vaccines for poultry. Third International Veterinary Vaccines and Diagnostics Conference, July 13-18, Guelph, Canada. ( Animal Health Reserch Reviews, 2004 - In Press).
- Tripathy, D.N. and Reed, W.M. 2003. Pox. In: Diseases of Poultry. 11th ed.,eds.Saif M. et al. Iowa State University Press, Ames, IA.
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Progress 01/01/03 to 12/31/03
Outputs
Genomic analysis of field strains of fowlpox virus reveals integration of reticuloendotheliosis virus (REV) in fowlpox virus genome. These viruses tend to persist in the poultry environment. The vaccine strains of fowlpox virus contain remnants of REV long terminal repeat (LTR) sequences. Since REV is widespread in poultry population, presence of REV LTR sequences in the genome of vaccine strains of fowlpox virus have potential to generate a fowlpox virus containing REV provirus. In this regard, immunization of birds with a fowlpox virus vaccine in which either all REV sequences have been deleted or only REV envelope gene is incorporated would avoid chances of emergence of a fowlpox virus which contains REV provirus. Since REV envelope gene has been associated with protection against REV, use of a fowlpox virus vaccine containing only REV envelope gene would provide protection against both fowlpox and REV. With a view to further evaluate the protective ability of two
genetically modified fowlpox viruses as vaccines against fowlpox, chickens were immunized either with genetically modified fowlpox virus lacking any REV sequences or with genetically engineered fowlpox virus containing REV envelope gene. To determine the protective ability of these vaccines, two weeks after immunization, the birds were challenged either with field strain of fowlpox virus, MN97 or PA97. All immunized birds developed localized lesions at the site of inoculation, which regressed between 3 to 4 weeks. Both vaccines provided protection against challenge with either of the two field strains of fowlpox virus.
Impacts
Persistent fowlpox virus in the poultry environment can be a source of infection for the susceptible birds. Immunization of birds with a fowlpox virus vaccine in which either all REV sequences have been deleted or only REV envelope gene is incorporated would provide protection against field strains of fowlpox virus. Further, application of such fowlpox virus vaccines would avoid chances of emergence of a fowlpox virus which contains REV provirus.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
1. Poxvirus isolate from ostriches is similar to fowlpox virus: An avian poxvirus was isolated from two 3-to 4-week old ostrich chicks. The ostrich chicks were raised on a previously poxvirus infected turkey premises. Antigenic (Western blotting), genetic (RFLP) and biologic (infection of susceptible chickens) studies did not reveal any significant difference between the poxvirus isolated from ostriches (PVO) and fowl poxvirus (FPV). Based upon our results and the history of disease outbreak, it is apparent that the persistent FPV in the poultry environment can be a source of infection for the susceptible birds. 2. A study on previously uncharacterized late promoters of FPV for inclusion in homologous recombinant vaccines: In an effort to characterize the late promoters of fowlpox virus (FPV), we identified few strong and weak promoters. Based on our preliminary studies, transcriptional regulatory elements controlling the expression of acidic type inclusion body
protein (ATI) gene and A15L vaccinia virus homologue dictated the most and the least amount of expressions respectively. To further understand the transcriptional regulation at molecular level, these two promoters were subjected to a series of mutational studies. Results of this study indicated that nucleotides immediately surrounding the transcriptional and translation start sites are highly critical to have a maximum level of expression. Based on this study, even the weakest transcriptional regulatory elements can be genetically manipulated to augment the level of expression. 3. Evaluation of Recombinant Vaccines for Protection against Fowlpox and Reticuloenotheliosis. Two recombinant fowlpox virus (FPV) vaccines generated to protect against either fowlpox or both fowlpox and reticuloendotheliosis were evaluated in chickens. The first recombinant FPV vaccine was generated by elimination of integrated reticuloendotheliois provirus (REV) sequences in the genome FPV. Another
recombinant FPV was generated in which all REV sequences from FPV were deleted and replaced with the gene encoding for the REV envelope protein. Responses of chickens immunized with either of these viruses, a commercial FPV vaccine as well as the birds of a control group were compared. The recombinant FPV generated by eliminating the REV provirus sequence showed lowered severity and persistence of lesions in chickens than the parent FPV although it provided protection against fowlpox. Chickens vaccinated with REV deleted FPV or the commercial vaccine were protected only against FPV challenge, but not against REV, while birds immunized with FPV containing REV envelope gene were protected against both viruses. Since the commercial FPV vaccines, which contain REV-LTR sequences, may have a potential for recombination with wild type REV, the recombinant vaccines generated in this study should be 'safer' when compared to the currently available commercial FPV vaccines.
Impacts
1. Persistent FPV in the poultry environment can be a source of infection for the susceptible birds. 2. Recombinant FPV(a) lacking all REV sequences and (b) containing REV envelope gene have been created. In preliminary evaluation, the former vaccine provides protection against fowlpox while the later provides protection against both REV and fowlpox. 3. Homologous fowlpox virus promoters can be genetically manipulated to create recombinant vaccines in which expression of foreign gene(s) is maximally optimized to create effective poultry vaccines.
Publications
- Shivprasad, H.L., Kim, T.-J. Woolcock, P.R. and Tripathy, D.N. 2002. Genetic and antigenic characterization of a poxvirus isolate from ostriches. Avian Dis. 46: 429-436.
- Tripathy, D.N. 2002. Future of new generation of virus-vectored vaccines for efficient poultry production. Proc. 51st Western Poultry Disease Conference (May 1 -4, 2002) Puerto Valletta, Mexico. Pp. 22-25.
- Srinivasan, V., Schnitzlein, W.M. and Tripathy, D.N. 2002. Infectivity and persistence of fowlpox virus. 5th Ann. Conf. on New and Re-emerging Infectious Diseases, University of Illinois, April 18-19, 2002. Abst. p 11.
- Srinivasan, V., Schnitzlein, W.M. and Tripathy, D.N. 2002. Role of CPD-photolyase and acidic-type inclusion body in maintaining the infectivity and environmental persistence of fowlpox virus. Poster Presentation at American Association of Avian Pathologists, American Veterinary Medical Association, Nashville, TN, July 14-17, 2002.
- Singh, P., Schnitzlein W.M. and Tripathy, D.N. 2002. Evaluation of a recombinant vaccine for protection against fowlpox in chickens. Poster Presentation (Best Poster Award) American Association of Avian Pathologists, American Veterinary Medical Association, Nashville, TN, July 14-17, 2002.
- Srinivasan, V., Schnitzlein, W.M. and Tripathy, D.N. 2002. Fowlpox virus encoded DNA repair enzyme, CPD-photolyase is involved in maintaining virus infectivity and persistence. Poster presentation, XIV International Poxvirus and Iridovirus Workshop, Lake Placid, NY, Sept. 20-25 P15, p 109.
- Singh, P. and Tripathy, D.N. 2002. Evaluation of recombinant vaccines for protection against fowlpox and reticuloendotheliosis. Poster presentation, XIV International Poxvirus and Iridovirus Workshop, Lake Placid, NY, Sept. 20-25, P63: p 157.
- Wang, X., Schnitzlein, W.M, Tripathy, D.N., Girshick T. and Khan, M.I. 2002. Construction and immunogenicity studies of recombinant fowlpox virus containing the S1 gene of Massachusetts 41 strain of IBV. Avian Dis. 46 (In Press).
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Progress 01/01/01 to 12/31/01
Outputs
Role of fowlpox virus-encoded photolyase in its pathogenesis. A comparative study of the response of eight-week old SPAFAS birds to cutaneous exposure to parental and photolyase-deficient fowlpox viruses revealed that the latter's replication was somewhat impaired. Nevertheless, this recombinant virus was still able to confer complete protection to the vaccinated chickens against a virulent fowlpox virus challenge. Role of A-type inclusion body protein in fowlpox virus. In fowlpox virus A-type inclusion body protein gene has apparently been duplicated and these two copies have genetically diverged to encode unrelated 55 and 70-kDa proteins. To study the role of A-type inclusions in fowlpox virus pathogenesis, the 70-kDa protein gene was insertionally inactivated. Since replication of the resultant recombinant in cultured cells was unaffected, A-type inclusion body protein is not required for virus multiplication. Homologue of a hemmagglutinin (HA)-like gene in fowlpox
virus genome. In order to determine the function of the protein encoded by the putative hemagglutination (HA) gene, we insertionally inactivated the respective ORF. Since no differences in the growth kinetics of the recombinant and parent virus were detected, the HA-like protein is not essential for virus replication in cultured cells. Physical characterization of the reticuloendotheliosis provirus in the genome of fowlpox virus. All field strains of fowlpox virus so far examined contain an avian reticuloendotheliosis provirus (7989 nucleotides in length) integrated at the same location in their genomes. In order to determine any genetic differences, the 3' long terminal repeat and the gp22 gene of the provirus associated with fourteen field strains of fowlpox virus were sequenced. These viruses were found to be genetically indistinguishable in regards to the above mentioned portion of provirus. Similarly, the provirus in the Australian fowlpox virus vaccine was identical to those of
the United States. Role of the integrated reticuloendotheliosis provirus in the pathogenesis of fowlpox. The genomes of all field strains of fowlpox virus have an integrated, nearly intact reticuloendotheliosis provirus. Additionally, each isolate is heterogeneous in that a small percentage of its constituents have retained one or the other of two variable lengths of ticuloendotheliosis virus LTR remnants in their DNAs. In contrast, each fowlpox virus vaccine strain is genetically homogeneous in that each one is composed entirely of viruses having only one of the two above mentioned LTR remnants.
Impacts
Several outbreaks of fowlpox virus have occured in previously vaccinated chicken flocks indicating re-emerging strains of fowlpox which may be antigenically different from vaccine viruses. In most cases, the diphtheritic form of disease with high mortality have been observed. The purpose of this project is to characterize these viruses antigenically, genetically and biologically.
Publications
- KIM, T.-J. and TRIPATHY, D.N. 2001. Reticuloendotheliosis virus integration in the fowl poxvirus genome: Not a recent event. Avian Diseases, 45: 663-669.
- SCHNITZLEIN, W.M., SINGH, P. and TRIPATHY, D.N. 2001. Genetic variability of reticuloendotheliosis provirus in the genome of fowlpox virus. PP No. 17. AAAP/AVMA Scientific Program, Boston, MA, P 53.
- SINGH, P., SCHNITZLEIN, W. and TRIPATHY, D.N. 2001. A caveat in FPV vaccines and their derived recombinants, a need for better vaccines. Abst. AAAP/AVMA Scientific Program, Boston, MA. P. 41.
- SRINIVASAN, V., SCHNITZLEIN, W. and TRIPATHY, D.N. 2001. Homologous fowlpox virus derived promoters for the development of recombinant vaccines. Abst. AAAP/AVMA Scientific Program, Boston, MA. P. 39.
- YU, L., LIU, W., SCHNITZLEIN, W.M., TRIPATHY, D.N. and KWANG, J. 2001. Study of protection by recombinant fowlpox virus expressing C-terminal nucleocapsid protein of infectious bronchitis virus against challenge. Avian Diseases 45:340-348.
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Progress 01/01/00 to 12/31/00
Outputs
Fowlpox virus (FPV) can persist in the poultry environment for extended periods. In this regard, we have identified two genes (a) Photolyase and (b) A-type inclusion in the genome of FPV, which appear to play an important role in its survival. The first encodes for a DNA repair enzyme, CPD-Photolyase. Since this enzyme repairs ultraviolet light damage to DNA by using white light as an energy source, our hypothesis is that its presence in FPV enhances the viability of this virus during its exposure to sunlight. Only a few members of the Poxviridae family produce A-type inclusions, which contain viral particles. We have identified two distinct, co-linear genes encoding putative proteins involved in the formation of A-type inclusion bodies. Although one of these gene product does not appear to be essential for viral replication, it is assumed that inclusion bodies probably shield the viral particles from environmental damage. We have also identified a putative
hemagglutin (HA) gene in the genome of all fowlpox viruses so far examined. Currently, the role or this gene in virus infectivity and/or pathogenicity is not known. Six homologous promoters, including a bi-drectional one, have also been identified in the genome of FPV. Their strengths are comparable to that of vaccinia virus promoters currently used for the creation of recombinant vaccines. These may have use in the development of a new generation of recombinant fowlpox virus, especially for use as polyvalent vaccines.
Impacts
Several outbreaks of fowlpox virus have occured in previously vaccinated chicken flocks indicating re-emerging strains of fowlpox which may be antigenically different from vaccine viruses. In most cases, the diphtheritic form of disease with high mortality have been observed. The purpose of this project is to characterize these viruses antigenically, genetically and biologically.
Publications
- Tripathy, D.N. 2000. Molecular techniques for the diagnosis of fowlpox. 137th AVMA Convention Notes., pp. 655-656.
- Srinivasan, V., Schnitzlein, W.M. and Tripathy, D.N. 2001. Fowlpox virus encodes a novel DNA repair enzyme, CPD-Photolyase, that restores infectivity of UV light-damaged virus. Journal of Virology, 75: 1681-1688.
- Srinivasan, V., Schnitzlein, W.M. and Tripathy, D.N. 2000. A novel repair enzyme expressed by fowlpox virus that restores the infectivity of UV-damaged virus. Poster Abst. P 51, XIIIth International Poxvirus and Iridovirus Symposium, Le Corum, Montpellier, France, p. 101.
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