Source: UNIVERSITY OF GEORGIA submitted to
ADVANCEMENTS IN THE ISOLATION, CHARACTERIZATION AND CONTROL OF AVIAN VIRUSES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
ANIMAL HEALTH
Reporting Frequency
Annual
Accession No.
0188756
Grant No.
(N/A)
Project No.
GEOV-0452
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2001
Project End Date
Sep 30, 2004
Grant Year
(N/A)
Project Director
Villegas, P. V.
Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
COLLEGE OF VETERINARY MEDICINE
Non Technical Summary
Viral diseases often present a detrimental economic effect upon the commercial poultry industry. The basis of this proposal is to test several viruses used to improve current vaccines agaiinst Newcastle and infectious bronchitis virus.. Avian adeno-associated viruses are harmless viruses for poultry that can be used to deliver antigens against different avian diseases. Infectious bronchitis vaccines should be tested to improve current efficacy of commercially available vaccines.
Animal Health Component
100%
Research Effort Categories
Basic
10%
Applied
40%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31132991101100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1101 - Virology;
Goals / Objectives
1. To continue testing the avian adeno associated virus vector to deliver genes for vaccine improvements. 2. To test several attenuated infectious bronchitis virus as vaccines.
Project Methods
1. The avian adeno associated virus with a Newcastle disease virus insert will be produced and tested in chickens. Immune response will be measured by the presence of antibodies against NDV. 2. The immuinogenicity of several infectious bronchitis virus will be tested by inoculating mature SPF chickens. Antibody detection will be done by the HI and VN tests.

Progress 10/01/01 to 09/30/04

Outputs
Avian Adeno-Associated Virus: Using a non-pathogenic, replication defective avian parvovirus, the avian adeno-associated virus (AAAV), we have generated a virus-based system for gene delivery in poultry. Aiming at this purpose we have cloned and sequenced the two known strains of the AAAV (VR-865 and DA-1). These infectious clones obtained were used to generate a plasmid-based system for the production We are currently using this plasmid-based system to generate recombinant AAAV coding for the VP2 gene of infectious bursal disease virus (Edgar strain) and the HN gene of the LaSota strain of Newcastle disease virus. This work is currently being funded by a grant from the US Poultry and Egg Association entitled "Use of the Avian Adeno-Associated Virus as a Vector for Poultry Vaccines". Infectious Bursal Disease: A heteroduplex mobility assay (HMA) was developed to genotype infectious bursal disease virus (IBDV). The HMA was able to differentiate standard, antigenic variants, and very virulent strains of IBDV. The results obtained by HMA where confirmed by RFLP and phylogenetic analysis of nucleotide sequences. The pathogenic properties of two IBDV isolates were compared by in vivo studies and in situ hybridization. The study included one standard (isolate 9109) and one Delaware variant strain (isolate 9865). Both strains induced subclinical disease, however by in situ hybridization some differences in the tissue tropism were observed Different molecular techniques such as reverse transcription-polymerase chain reaction / restriction fragment length polymorphism (RT-PCR/RFLP), heteroduplex mobility assay (HMA), nucleotide and amino acid sequence analysis, and riboprobe in situ hybridization (ISH) were used to perform the genetic and pathological characterization of field strains of IBDV. From the samples analyzed from the United States, 80% exhibited RFLP identical to the variant Delaware E strain.

Impacts
Improvements in the methodology used to identify field isolates of Infectious Bursal Disease Virus.

Publications

  • Banda A, Villegas P, El-Attrache J. Heteroduplex mobility assay for genotyping infectious bursal disease virus. Avian Dis. 2004 Dec;48(4):851-62.
  • Banda A, Villegas P. Genetic characterization of very virulent infectious bursal disease viruses from Latin America. Avian Dis. 2004 Sep;48(3):540-9.
  • Estevez C, Villegas P. Sequence analysis, viral rescue from infectious clones and generation of recombinant virions of the avian adeno-associated virus. Virus Res. 2004 Oct;105(2):195-208.
  • Estevez C, Villegas P, El-Attrache J. A recombination event, induced in ovo, between a low passage infectious bronchitis virus field isolate and a highly embryo adapted vaccine strain. Avian Dis. 2003 Oct-Dec;47(4):1282-90.
  • Banda A, Villegas P, El-Attrache J. Molecular characterization of infectious bursal disease virus from commercial poultry in the United States and Latin America. Avian Dis. 2003 Jan-Mar;47(1):87-95.
  • Garcia M, El-Attrache J, Riblet SM, Lunge VR, Fonseca AS, Villegas P, Ikuta N. Development and application of reverse transcriptase nested polymerase chain reaction test for the detection of exogenous avian leukosis virus. Avian Dis. 2003 Jan-Mar;47(1):41-53.
  • Zavala G, Dufour-Zavala L, Villegas P, El-Attrache J, Hilt DA, Jackwood MW. Lack of interaction between avian leukosis virus subgroup J and fowl adenovirus (FAV) in FAV-antibody-positive chickens. Avian Dis. 2002 Oct-Dec;46(4):979-84.
  • Majo N, El-Attrache J, Banda A, Villegas P, Ramis A, Pages A, Ikuta N. Molecular characterization of Spanish infectious bursal disease virus field isolates. Avian Dis. 2002 Oct-Dec;46(4):859-68.
  • Alvarado IR, Villegas P, El-Attrache J, Brown TP. Evaluation of the protection conferred by commercial vaccines against the California 99 isolate of infectious bronchitis virus. Avian Dis. 2003 Oct-Dec;47(4):1298-304.


Progress 01/01/03 to 12/31/03

Outputs
Several avian viruses have been tested as vectors to deliver different genes to immunize chickens against several diseases. The avian adeno associated viruses (AAAV) can be safely used for in-ovo inoculation, widely used procedure in the US poultry industry. A plasmid-based system to generate recombinant AAAV coding for immunogenic proteins derived from Newcastle disease, avian influenza and infectious bursal disease viruses, is being developed and tested for protection studies in-vivo. Several strains of infectious bronchitis virus (IBV) have been adapted to grow in different systems in an attempt to decrease their pathogenicity for the respiratory tract. One Arkansas serotype strain has been tested in chickens and found that it does still replicates in the upper respiratory tract of chickens, although inducing significantly less reaction than a commercial vaccine.

Impacts
Improvements in vaccination efficiency and decrease in post vaccination reactions that will result in increase productivity.

Publications

  • Estevez, Carlos, P. Villegas, and J. El-Attrache. A Recombination Event, Induced in ovo, Between a Low Passage Infectious Bronchitis Virus Field Isolate and a Highly Embryo Adapted Vaccine Strain. Avian Dis. 47:1282-1290. 2003.
  • Alvarado, I. R., P. Villegas, J. El-Attrache, and T. P. Brown. Evaluation of the Protection Conferred by Commercial Vaccines Against the California 99 Isolate of Infectious Bronchitis Virus. Avian Dis. 47:1298-1304. 2003.


Progress 01/01/02 to 12/31/02

Outputs
Use of the Avian Adeno-Associated Virus as a Vector for Gene Delivery In the poultry industry, recombinant viruses have been used for the expression of immunogenic peptides in the field. Disadvantages related to the use of recombinant viruses include the development of immunity against the viral vector, activation of oncogenes and restriction of introduction of genes of interest to the tissues that are permissive to the viral vector. In recent years, extensive work has been done in the characterization and use of the human adeno associated virus as a viral vector for gene delivery. The advantages of the adeno associated virus as vectors include the lack of pathogenicity of the virus, the ability to infect a wide variety of tissues and cells, the ability to accommodate relatively large pieces of genomic information and the ability to integrate in a non-random fashion into the host's genome without causing integrational activation of oncogenes. The purpose of our current research is to use the avian adeno associated virus as a viral vector for the introduction and expression of immunogenic peptides in vivo as a mean to induce a protective immune response against disease. Adaptation of Infectious bronchitis virus (IBV) to intestinal tissues IBV vaccination in poultry results in post vaccination reaction characterized respiratory signs than when complicated result in severe economic losses. Therefore, we are trying to grow IBV to the intestinal tissue of chickens in an attempt to adapt this virus to this tissue, thus reducing the post vaccinal respiratory reaction commonly observed. Viruses have been grown in intestinal explants and have been recovered from the intestinal tract of chickens. Preliminary pathogenicity tests performed in commercial chickens have shown that further attenuation is necessary to decrease the respiratory tropism of this virus.

Impacts
Avian adeno-associated viruses are harmless viruses for poultry that can be used to deliver antigens against different avian diseases.

Publications

  • Majo, N., J. El-Attrache, A. Banda, P. Villegas, A. Ramis, A. Pages, and N. Ikuta. 2002. Molecular characterization of Spanish Infectious Bursal Disease Virus field isolates. Avian Dis. 46:859-868.
  • Zavala, G., L. Dufour-Zavala, P. Villegas, J. El-Attrache, D.A. Hilt, and M.W. Jackwood. 2002. Research Note-Lack of interaction between avian leucosis virus subgroup J and fowl adenovirus (FAV) in FAV-antibody-positive chickens. Avian Dis. 46:979-984.
  • Garcia, Maricarmen, J. El-Attrache, S.M. Riblet, V.R. Lunge, A.S.K. Fonseca, P. Villegas, and N. Ikuta. 2003. Development and application of reverse transcriptase nested polymerase chain reaction test for the detection of exogenous avian leukosis virus. Avian Dis. 47:41-53.
  • Banda, A., P. Villegas, and J. El-Attrache. 2003. Molecular characterization of Infectious Bursal Disease Virus from commercial poultry in the United States and Latin America. Avian Dis. 47:87-95.


Progress 01/01/01 to 12/31/01

Outputs
Specific research involving infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV) and avian adenovirus along with concluded research on avian leukosis virus subgroup J (ALV-J) are the focus of this continuation proposal. IBV recombination potential was characterized in vitro by RT-PCR and sequence analysis between the Massachusetts and the Delaware 072-like strain. Separately, adaptation of vaccine and field strains of IBV to replicate in the intestinal tract of chickens, have been performed via serial passages in SPF chickens. Procedures have been established to create and maintain intestinal ring tissue culture and utilized to further propagate these strains. Challenge studies will be performed and selected vaccine candidates will be evaluated. Variants of IBDV have been characterized in our laboratory via RT-PCR/RFLP, heteroduplex mobility assays (HMA), sequence analysis and in vivo pathological studies. In addition, selected chicken anemia virus (CAV) field isolates have also been characterized via sequence and clinical analysis. Molecular and clinical analysis of these isolates have precluded future characterization of CAV and IBDV co-infection via in situ hybridization techniques. Selected pathogenic avian adenovirus field isolates have been characterized via molecular and clinical methods. Serological responses induced by theses isolates will be assayed by an avian adenovirus ELISA that is utilized by a US primary breeder and compared with the virus neutralization assay. Classical and molecular studies involving ALV-J were completed. These studies included comparative analysis of different cell cultures for viral propagation, identification of RT-PCR ALV-J positive samples undetected by virus isolation and the design and evaluation of antisense oligos as potential inhibitors of ALV-J replication.

Impacts
Pathogenic viruses, which present a continual hazard to the commercial poultry industry, are the basis of our current research. Specifically, diseases caused by infectious bronchitis virus (IBV), avian leukosis virus subgroup J (ALV-J) and infectious bursal disease virus (IBDV) are the focus of this laboratory's ongoing research. The protection provided by currently available commercial IBV vaccines is not as satisfactory as expected. Therefore, challenge studies are conducted and evaluated in order to develop products that provide better protection against IBV. ALV-J has been reported to play a role in increasing condemnations at processing plants, mortality with or without tumors, a decrease in egg production and depletion of chick uniformity are some of the effects caused of this virus. Presently, virus isolation in specific cell culture along with the use of molecular techniques such as RT-PCR and sequence analysis are being used to generate data for progressive research on ALV-J pathogenicity and oncogenicity. IBDV is an acute and highly transmissible virus, which can cause clinical disease and mortality along with immunosuppresion in young chickens. Molecular analysis is used to rapidly identify and classify IBDV strains. This has led to the recent discovery and identification of a very virulent strain of IBDV for the first time in the American continent. Monitoring of IBDV field isolates allows for modification of vaccination procedures in order to provide improved protection against pathogenic viruses.

Publications

  • Banda, A., P. Villegas, J. El-Attrache, and H. Sellers. The use of the heteroduplex mobility analysis (HMA) for genotyping field isolates of the Infectious Bursal Disease Virus (IBDV). Proc. of Southern Conference on Avian Diseases, World Congress Center, Atlanta, GA. (Abstract No. 229, pp. 51-52). January 14-15, 2002.
  • Estevez, C.N., and P. Villegas. Sequence analysis of the avian adeno associated virus. Proc. of Southern Conference on Avian Diseases, World Congress Center, Atlanta, GA. (Abstract No. 241, p. 54). January 14-15, 2002. (Received The C. S. Eidson Award for BEST POSTER.)
  • Majo, N., J. El-Attrache, A. Banda, P. Villegas, A. Ramis, A. Pages, and N. Ikuta. Molecular characterization and identification of Spanish Infectious Bursal Disease Virus field isolates. Avian Dis. 2002.
  • Alvardo, Ivan, P. Villegas, J. El-Attrache, and J. Glisson. Protection afforded by commercial vaccines against the Nebraska 95 IBV field isolate. Proc. of the 138th Annual Meeting of the American Veterinary Medical Association, Boston, MA. July 14-18, 2001.
  • Banda, A., P. Villegas, J. El-Attrache, and C. Brown. Pathogenesis of Chicken Infectious Anemia virus: studies on latency. Proc. of the 138th Annual Meeting of the American Veterinary Medical Association, Boston, MA. July 14-18, 2001.
  • Ikuta, N., J. El-Attrache, P. Villegas, M. Garcia, V.R. Lunge, A.S.K. Fonseca, C. Oliveira, and E.K. Marques. Molecular characterization of Brazilian infectious bursal disease viruses. Avian Dis. 45:297-306. 2001.
  • El-Attrache, J., P. Villegas, B. O'Connor, J.R. Buhr, and G.N. Rowland. Adenovirus pathogenicity in immature ostriches. Avian Dis. 45:442-446. 2001.
  • Banda, A., Villegas, P., El-Attrache, J., and Estevez, C.: Molecular characterization of seven field isolates of infectious bursal disease virus obtained from commercial broiler chickens. Avian Dis. 45:620-630. 2001.
  • El-Attrache, John, and Pedro Villegas. Genomic identification and characterization of avian adenoviruses associated with inclusion body hepatitis. Avian Dis. 45:780-787. 2001.
  • Ruano, Miguel, J. El-Attrache, and Pedro Villegas. Efficacy comparison of disinfectants used by the commercial poultry industry. Avian Dis. 45:972-977. 2001.
  • El-Attrache, John, Pedro Villegas, and Maricarmen Garcia. Classical and molecular analysis of nested RT-PCR ALV-J positive plasma samples undetected by virus isolation coupled with antigen capture ELISA. Proceedings of the 138th Annual Meeting of the American Veterinary Medical Association, Boston, MA. July 14-18, 2001.
  • Villegas, P., M. Ruano, J. El-Attrache, and N. Ferguson. Virological and serological identification of Infectious Bronchitis Virus (IBV) isolates known as DE-072 variants. Proc. of the 138th Annual Meeting of the American Veterinary Medical Association, Boston, MA. July 14-18, 2001.
  • Banda, A. and P. Villegas. Deteccion y tipificacion de cepas del virus de la enfermedad infecciosa de la bolsa mediante tecnicas moleculares. (Detection and classification of infectious bursal disease virus by molecular techniques.) Proc. of the 3rd International Seminar - Boehringer-Ingelheim, Guadalajara, Mexico. Pp. 9-20. October, 2001.
  • Villegas, Pedro. Enfermedad infecciosa de la bolsa: Impacto y estrategias de control. (Infectious bursal disease: Impact and control strategies.) Proc. of the 3rd International Seminar - Boehringer-Ingelheim, Guadalajara, Mexico. Pp. 21-27 Ocrober, 2001.
  • Alvarado, Ivan, Pedro Villegas, John El-Attrache, and Mark Jackwood. Persistence of Infectious Bursal Disease (IBV) Arkansas and Massachusetts vaccines in tissues from broilers. Proc. of Southern Conference on Avian Diseases, World Congress Center, Atlanta, GA. (Abstract No. 225, p. 51). January 14-15, 2002. (Received The C. S. Eidson Award for BEST ORAL PRESENTATION.)