Source: TEXAS A&M UNIVERSITY submitted to
ROLE OF THE HEMAGGLUTININ PROTEIN (HA) IN THE PATHOGENESIS OF A MODERATELY PATHOGENIC H5N3 AVIAN INFLUENZA VIRUS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
ANIMAL HEALTH
Reporting Frequency
Annual
Accession No.
0211434
Grant No.
(N/A)
Project No.
TEX09244
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 3, 2007
Project End Date
Sep 30, 2009
Grant Year
(N/A)
Project Director
Lupiani, B.
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
VETERINARY PATHOBIOLOGY
Non Technical Summary
Avian influenza (AI) is a major respiratory disease of poultry that has the potential to cause catastrophic losses to the commercial poultry industry worldwide. The overall objective of this application is to investigate the role of the hemagglutinin of moderately pathogenic (H5N3) and non-pathogenic (H5N2) AI virus on pathogenesis in chickens.
Animal Health Component
100%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
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
Objective 1. Generation of wild type and mutant avian influenza viruses using reverse genetics. We will use the experimental approach of generating recombinant avian influenza viruses in which the hemagglutinin gene from a moderately pathogenic (H5N3) AI virus will be transferred to a non-pathogenic virus (H5N2) and vice versa. In addition sequences with potential pathogenic potential will be removed from H5N3 and introduced into H5N2 to determine their role in pathogenesis. Objective 2. In vivo characterization of AI mutant viruses. We will test the working hypothesis that amino acids differences at the cleavage site of the hemagglutinin gene of H5N3 and H5N2 are responsible for differences in pathogenicity between these two viruses. We will test this hypothesis by inoculating chickens with wild type and mutant AI viruses generated by reverse genetics.
Project Methods
Objective 1. Generation of wild type and mutant avian influenza viruses using reverse genetics. The objective of this section is to generate wild type and mutant AI viruses in order to determine the role of the hemagglutinin gene in the pathogenesis of H5N3 virus. Generation of mutant hemagglutinin clones. The cleavage site of H5 from H5N2 and H5N3 will be mutated to contain one extra and one less basic amino acid, respectively, using standard site directed mutagenesis techniques. The presence of the desired mutations will be confirmed by DNA sequencing of these constructs. From the proposed studies we expect to identify the effect of basic amino acids in the cleavage site of the hemagglutinin gene in pathogenesis. Generation of recombinant viruses. Recombinant rH5N3, rH5N2, rH5N3-HA, rH5N2-HA, rH5N3-DHA and rH5N2+HA will be generated by transfecting the wild type and/or mutant plasmids containing the 8 genes of AI virus into 293T-CEF co-cultures. Recovered viruses will be collected and used to inoculate 10-day-old embryonated chicken eggs by the allantoic route. The titer of the viruses generated will be determined in embryonated chicken eggs and calculated as EID50/ml. Objective 2. In vivo characterization of AI mutant viruses Pathogenesis of wild type and mutant viruses. Wild type and mutant AI viruses will be inoculated into 1 day and 4-week-old chickens to evaluate specific biological properties such as replication, shedding, and pathogenesis. Pathogenesis studies in 1 day-old chickens. After hatch, SPF chickens will be randomly divided into 8 groups (12 chickens per group) and inoculated intravenously (4 groups) or by the intrachoanal cleft route (4 groups) with 107 EID 50 of wild type and mutant viruses. Chicks inoculated with allantoic fluid of uninfected embryonated chicken eggs will be used as negative controls. Chickens will be observed daily for clinical signs of disease for a total of 7 days. Six chickens from each group will be euthanized at 4 and 7 days post inoculation (PI) and tissue samples taken for antibody response, histological and immunohistochemistry analysis and virus titration. Pathogenesis studies in 4 week-old chickens. After hatch, chickens will be randomly divided into 4 groups (12 chickens per group). At 4 weeks of age, each group will be inoculated with 0.2 ml/chicken containing 107 EID 50 of the wild type and mutant viruses by the intrachoanal cleft routes. Chickens inoculated with allantoic fluid of uninfected embryonated chicken eggs will be used as negative controls. Chickens will be observed daily for clinical signs of disease for a total of 7 days. Six chickens in each group will be euthanized at 4 and 7 days PI and tissue samples taken for antibody response, histological and immunohistochemistry analysis and virus titration.

Progress 07/03/07 to 09/30/09

Outputs
OUTPUTS: Using a reverse genetic system developed in our laboratory, we generated 6 recombinant AIV: parental non-pathogenic (AI-102); parental mildly pathogenic (AI-103); HA and NA from H5N2 all other genes from H5N3 (AI-113); HA and NA from H5N3 all other genes from H5N2 (AI-116); HA H5N3 all other genes from H5N2 (AI-118); HA from H5N2 all other genes from H5N3 (AI-119). After recovery of the viruses in cell culture, viral stocks were generated in 10-day-old embryonated chicken eggs and the replication properties determined. As seen in Table 1, no significant differences in virus titers were observed among the recombinant viruses generated suggesting that exchange of HA or HA and NA genes among viruses does not affect virus replication in embryonated chicken eggs. The role of HA or HA and NA in virus pathogenesis was evaluated by inoculating 4 week-old chickens with 0.1 ml/chicken containing 107 EID 50 of parental and mutant viruses by the intrachoanal cleft routes. Chickens inoculated with allantoic fluid of uninfected embryonated chicken eggs were used as negative controls. Chickens were observed daily for clinical signs of disease for a total of 7 days. Four chickens in each group were euthanized at 4 and 7 days PI and examined for gross lessions. Prior to euthanasia at 7 DPI, chickens were bled and presence of antibodies to AIV determined by ELISA. Our results show that the pathogenicity of the viruses correlated with the type of HA and NA present. The viruses with the HA protein from H5N3 were moderately pathogenic while those with HA from H5N2 were non-pathogenic. Although no clinical signs of disease were observed, gross lesions upon necropsy included tracheal plugs and hemorrhagic trachea and lungs. No significant differences in antibody response were observed among any of the viruses tested. PARTICIPANTS: Sanjay Reddy, Associate Professor, Texas A&M University Vinayak Brahmaksatriya, Graduate Student, Texas A&M University Edu Suarez-Martinez, Associate Professor, University of Puerto Rico, Ponce Noried DeJesus-Velazquez, Undergraduate Student, University of Puerto Rico, Ponce TARGET AUDIENCES: Researchers, students and the poultry industry through laboratory instruction and scientific presentations PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
New avian influenza viruses from low pathogenic strains have been generated by reverse genetics. Our in vivo studies confirm that the hemmaglutinin protein plays an important role in avian influenza pathogenesis in chickens. In addition, deep sequencing of RNA isolated from wild type avian influenza virus infected chickens revealed the presence of differentially expressed micro RNAs which may play a role in virus replication and pathogenicity.

Publications

  • De Jesus-Velazquez N.M., Suarez-Martinez E.B. and Lupiani B. 2007. Understanding avian influenza virus: infection, pathogenicity and prevalence. Briefing of MSI interns to DHS Under Secretary Jay M. Cohen and other key S&T officials. August 2007, Washington DC.
  • Wang Y., Brahmakshatriya V., Zhu H., Lupiani B., Reddy S.M., Yoon B-J, Gunaratne P.H., Kim J.H., Chen R., Wang J., Zhou H. 2009. Identification of differentially expressed miRNAs in chicken lung and trachea with avian influenza virus infection by a deep sequencing approach. BMC Genomics 10 (512) doi:10.1186/1471-2164-10-512


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Using a non-pathogenic (H5N2) and a moderately (H5N3) avian influenza viruses we have generated mutant and chimera viruses in which the hemagglutinin (HA) or neuraminidase (NA) proteins have been modified. The replication properties of these viruses were evaluated in embryonated chicken eggs and no differences in virus titers were observed among the wild type and mutant viruses. Parental and recombinant viruses were inoculated in chickens and their pathogenicity determined. Our results show that the pathogenicity of the viruses correlated with the presence the type of HA and NA present. All the viruses with the H5N3 combination were moderately pathogenic while the ones with H5N2 combination were non-pathogenic. PARTICIPANTS: Sanjay Reddy, Associate Professor, Texas A&M University Vinayak Brahmaksatriya, Graduate Student, Texas A&M University Edu Suarez-Martinez, Associate Professor, University of Puerto Rico, Ponce Noried De Jesus-Velazquez, Undergraduate Student, University of Puerto Rico, Ponce TARGET AUDIENCES: The target audience for this project is the scientific community, as better understanding on the pathogenesis of avian influenza viruses in chickens may lead to the development of improved vaccines and control measurements. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
New avian influenza viruses from low pathogenic strains have been generated by reverse genetics. Our in vivo studies confirm that the hemmaglutinine protein plays an important role in avian influenza pathogenesis in chickens.

Publications

  • De Jesus-Velazquez N.M., Suarez-Martinez E.B. and Lupiani B. Understanding avian influenza virus: infection, pathogenicity and prevalence. Annual Biomedical Research Conference for Minority Students (ABRCMS) November 7-10, 2007 in Austin, TX


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Using a non-pathogenic (H5N2) and a moderately (H5N3) avian influenza viruses we have generated mutant and chimera viruses in which the hemagglutinin (HA) or neuraminidase (NA) proteins have been modified. The replication properties of these viruses were evaluated in embryonated chicken eggs and no differences in virus titers were observed among the wild type and mutant viruses. The next step in this project will be to compare the pathogenicity of wild type and mutant viruses in chickens to determine the role of the HA and NA proteins, individualy and in combination, in the pathogenicity of these viruses. PARTICIPANTS: Sanjay Reddy, Associate Professor, Texas A&M University Vinayak Brahmaksatriya, Graduate Student, Texas A&M University Edu Suarez, Associate Professor, University of Puerto Rico, Ponce Noried de Jesus, Undergraduate Student, University of Puerto Rico, Ponce TARGET AUDIENCES: The target audience for this project is the scientific community, as better understanding on the pathogenesis of avian influenza viruses in chickens may lead to the development of improved vaccines and control measurements. PROJECT MODIFICATIONS: None

Impacts
New avian influenza viruses from low pathogenic strains have been generated by reverse genetics. These viruses will provide invaluable information on the role of the HA and NA proteins in the pathogenesis of these viruses.

Publications

  • No publications reported this period