The Immunopathogenesis of Avian Influenza Virus in Avian Species

NON-TECHNICAL SUMMARY: Avian influenza is one of the most important diseases of poultry.

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APPROACH: <ol>
<li> Microarray analysis: The chicken HTC macrophage cell line and primary macrophages prepared from chickens and ducks will be infected with A/pheasant/CA/2373/1998 (H9N2), A/chicken/CA/1772/2002 (H6N2), A/turkey/TX/39/97 (H9N2) and A/chicken/CA/6754/02 (H6N2). These four viruses have been selected because they have NS1 genes that belong to the A and B alleles, respectively. We anticipate that NS1 mediates the effects of AIV on macrophages and would like to test viruses with different NS1 alleles to determine if it mediates the downregulation of immune function genes as we described in the Prelimiary Results. Primary monocytes/macrophages will be collected from chickens and ducks and cultured in RPMI/1640 medium. 5x106 cells will be plated on 10-cm tissue culture plates 16 hrs before infection. In a one-cycle infection, the cells will be infected at a multiplicity of infection (moi) of 1 and incubated at 37C. Total RNA will be prepared and microarray reactions will be accomplished following standard Affymetrix protocols. cRNA probe hybridization to Chicken GeneChips (Affymetrix, Santa Clara, CA) and scanning will be performed at the Genome Center, UC Davis following the Affymetrix Manual. <li> The cells will be observed every 6 hours for cytopathic effects. The cultures will be collected for apoptosis analysis and RNA extraction. Additionally, at 6 and 12 hours post infection, the cells will be fixed, and an immunofluorescent antibody assay will be performed to detect AIV. Cultures will be grown in triplicate and realtime RT-PCR will be used on individual cultures to confirm the microarray findings on selected genes, which will initially include: MHC class I, MHC class II, MHC B-G, IL-1b, IL-4, and CCL5. This list may be expanded based on subsequent findings. <li> Reverse genetics: NS1 genes will be altered through site mutagenesis and mutated viruses will be generated through co-transfection of macropahges with a reverse genetics system to determine which part of the NS1 genes is responsible for the observed differences in cytopathogenic effect (apoptosis) and the modulation of the immnue genes detected by microarray analysis. <li> Yeast two-hybrid system: A genetic apprach using the yeast two-hybrid system will be employed to detect the host interacting protein(s) in macrophages which interacts with the NS1. Cell biology approaches will next be used to determine the consequence of the interaction in the viral infection, cell signaling pathways and the host pathogenesis both in macrophages and in vivo. </OL>
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PROGRESS: 2007/01 TO 2007/12<BR>
OUTPUTS: We have made the following progresses which are related to the goals and objectives since the project started: <BR>
1. Extensive Regulations of immune genes in chicken macrophages and lungs infected with influenza virus H9N2. Pro-inflammatory cytokines IL-1beta, IL-8, and chemokines K203, ah221 (CCL7), ah294, CCL3, CCL20, K60 (CXCL1) and CXCL14 were all upregulated from 1.3 to 26.7 fold in A/ph/CA/2373/98 (H9N2) virus infected Macrophages. However, cytokines IL-2, IL-6, IL-16, IL-17 as well as IL-4 receptor(IL-4R) and IL-21R were downregulated between 1.2 and 10.8 fold. We also found that interferon (IFN) and IFN-inducible genes were regulated differentially in A/ph/CA/2373/98 (H9N2) virus infected Macrophages. While 2'-5'-oligoadenylate synthetase (OAS)-like gene was upregulated at the 6th hour post infection up to 3.3 fold, Mx protein was downregulated marginally. Expression of IFN-inducible protein IP-30 was suppressed up to 2.4 fold but that of interferon-inducible 58 kDa with tetratricopeptide repeats 5 (IFIT-5) was increased up to 20.1 fold in the early stage of infection (6 hrs post infection). IFN beta and gamma were both expressed at low levels in control Macrophages. While the IFN beta expression was increased up to 1.7 fold 12 hrs post infection, interferon gamma expression did not change. <BR> <BR> 2. Characterize the impact of immune modulation by LPAI viruses on host immune responses. We examined how the host adaptive immunity developed in both H6N2 and H9N2 virus-infected chickens. Swabs from 8 of 9 chickens infected with A/ch/CA/1772/02 (H6N2) had virus detectable by real time RT-PCR post inoculation through day 12. Within two weeks post infection, the Hemagglutination inhibition (HI) antibodies titers ranged from 1:20 to 1:160 and the seroconversion rate was 88.9% (8/9). In contrast, 8 of 9 chickens challenged with H9N2 shed viruses post inoculation through day 12 as detected by real-time RT-PCR, but all of the infected birds showed HI antibody titers less than 1:20 and the seroconversion rate was 0% (0/9) two weeks post infection, indicating that the adaptive immune responses were suppreseed in H9N2 infected chickens. All challenged birds survived infection. Necropsy of the hens showed that the H9N2 virus infected hens had no grossly detectable lesions while some of the H6N2 virus infected hens had mild lesions in the respiratory tract. <BR> <BR> 3. Characterization of the viral genes that regulate host immune responses. We proposed to use reverse genetics apporach to study the roles of the viral genes that regulate host immune responses. We targeted NS1 gene of the H9N2 virus. At this stage we have cloned all eight segments of genomic cDNA into the vector pDP2000, transfected them in MDCK/293T cells and obtained rescued viruses which were infectious and could be passaged in cell cultures and embryonated eggs. We have also generated mutant NS1 gene and rescued the recombinant viruses which lacks in NS1. We will use the rescued wild-type and NS1-mutant viruses for infection in both chicken macrophages and chickens. The regulated expression of immune genes and the regulation of the adaptive immune responses in infected chickens will be examined. <BR> PARTICIPANTS: Xing, Z.: Department of Pathology, Microbiology & Immunology, School of Veterinary Medicine, UC Davis Cardona, C.J.: Department of Population Health & Reproduction, School of Veterinary Medicine, UC Davis Li, J.L.: Department of Population Health & Reproduction, School of Veterinary Medicine, UC Davis Dao, N.: Department of Population Health & Reproduction, School of Veterinary Medicine, UC Davis <BR> TARGET AUDIENCES: Researchers and managers in poultry industry; Health professionals in veterinary and human infectious diseases; <BR> PROJECT MODIFICATIONS: No major changes are taken at this stage.

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IMPACT: 2007/01 TO 2007/12<BR>
1. Regulated expression of immune genes in influenza virus-infected mammals have been extensively reported but our data were the first to report the immune responses in chicken macrophages responding to influenza virus infection. Our findings demonstrated that a correlation exists between the regulated gene expression in infected macrophages and the suppression of the antibody responses. This indicates that chicken macrophages could play a significant roles in both innate and adaptive immune responses in chickens infected with influenza viruses. <BR> <BR> 2. The virus strain, A/ph/CA/2373/98 (H9N2), caused extensive downregulation of immune genes and poor antibody responses in chickens. This fact showed the importance of the selection of candidate viral strains for live vaccine development. The viral strains, selected for live vaccine candidates, should have least suppressive impact on the the host immune responses.

<p>

PROGRESS: 2007/01/01 TO 2007/12/31<BR>
OUTPUTS: We have made the following progresses which are related to the goals and objectives since the project started: 1. Extensive Regulations of immune genes in chicken macrophages and lungs infected with influenza virus H9N2. Pro-inflammatory cytokines IL-1beta, IL-8, and chemokines K203, ah221 (CCL7), ah294, CCL3, CCL20, K60 (CXCL1) and CXCL14 were all upregulated from 1.3 to 26.7 fold in A/ph/CA/2373/98 (H9N2) virus infected Macrophages. However, cytokines IL-2, IL-6, IL-16, IL-17 as well as IL-4 receptor(IL-4R) and IL-21R were downregulated between 1.2 and 10.8 fold. We also found that interferon (IFN) and IFN-inducible genes were regulated differentially in A/ph/CA/2373/98 (H9N2) virus infected Macrophages. While 2'-5'-oligoadenylate synthetase (OAS)-like gene was upregulated at the 6th hour post infection up to 3.3 fold, Mx protein was downregulated marginally. Expression of IFN-inducible protein IP-30 was suppressed up to 2.4 fold but that of interferon-inducible 58 kDa with tetratricopeptide repeats 5 (IFIT-5) was increased up to 20.1 fold in the early stage of infection (6 hrs post infection). IFN beta and gamma were both expressed at low levels in control Macrophages. While the IFN beta expression was increased up to 1.7 fold 12 hrs post infection, interferon gamma expression did not change. 2. Characterize the impact of immune modulation by LPAI viruses on host immune responses. We examined how the host adaptive immunity developed in both H6N2 and H9N2 virus-infected chickens. Swabs from 8 of 9 chickens infected with A/ch/CA/1772/02 (H6N2) had virus detectable by real time RT-PCR post inoculation through day 12. Within two weeks post infection, the Hemagglutination inhibition (HI) antibodies titers ranged from 1:20 to 1:160 and the seroconversion rate was 88.9% (8/9). In contrast, 8 of 9 chickens challenged with H9N2 shed viruses post inoculation through day 12 as detected by real-time RT-PCR, but all of the infected birds showed HI antibody titers less than 1:20 and the seroconversion rate was 0% (0/9) two weeks post infection, indicating that the adaptive immune responses were suppreseed in H9N2 infected chickens. All challenged birds survived infection. Necropsy of the hens showed that the H9N2 virus infected hens had no grossly detectable lesions while some of the H6N2 virus infected hens had mild lesions in the respiratory tract. 3. Characterization of the viral genes that regulate host immune responses. We proposed to use reverse genetics apporach to study the roles of the viral genes that regulate host immune responses. We targeted NS1 gene of the H9N2 virus. At this stage we have cloned all eight segments of genomic cDNA into the vector pDP2000, transfected them in MDCK/293T cells and obtained rescued viruses which were infectious and could be passaged in cell cultures and embryonated eggs. We have also generated mutant NS1 gene and rescued the recombinant viruses which lacks in NS1. We will use the rescued wild-type and NS1-mutant viruses for infection in both chicken macrophages and chickens. The regulated expression of immune genes and the regulation of the adaptive immune responses in infected chickens will be examined. <BR> PARTICIPANTS: Xing, Z.: Department of Pathology, Microbiology & Immunology, School of Veterinary Medicine, UC Davis Cardona, C.J.: Department of Population Health & Reproduction, School of Veterinary Medicine, UC Davis Li, J.L.: Department of Population Health & Reproduction, School of Veterinary Medicine, UC Davis Dao, N.: Department of Population Health & Reproduction, School of Veterinary Medicine, UC Davis <BR> TARGET AUDIENCES: Researchers and managers in poultry industry; Health professionals in veterinary and human infectious diseases; <BR> PROJECT MODIFICATIONS: No major changes are taken at this stage.

<p>

IMPACT: 2007/01/01 TO 2007/12/31<BR>
1. Regulated expression of immune genes in influenza virus-infected mammals have been extensively reported but our data were the first to report the immune responses in chicken macrophages responding to influenza virus infection. Our findings demonstrated that a correlation exists between the regulated gene expression in infected macrophages and the suppression of the antibody responses. This indicates that chicken macrophages could play a significant roles in both innate and adaptive immune responses in chickens infected with influenza viruses. <BR> <BR> 2. The virus strain, A/ph/CA/2373/98 (H9N2), caused extensive downregulation of immune genes and poor antibody responses in chickens. This fact showed the importance of the selection of candidate viral strains for live vaccine development. The viral strains, selected for live vaccine candidates, should have least suppressive impact on the the host immune responses.