Regulation of Host Responses and Viral Replication in Highly Pathogenic Avian Influenza H5N1-Infected Chickens

NON-TECHNICAL SUMMARY: The H5N1 highly pathogenic influenza A viruses remain a major global concern because of their rapid evolution, ongoing circulation in wild and domestic birds, and potential to transmit among humans. Little has been known about the pathogenesis of HPAI infection in chickens. We have previously demonstrated the induction of proinflammatory cytokines and virus-induced apoptosis in immune cells and respiratory tissues, which may contribute to high pathogenicity in birds. However, the mechanisms to regulate the host response and the significance of host-viral interactions in H5N1-infected birds remain poorly understood. In this project we will examine the roles of MAP kinases in the regulation of immune responses in HPAI H5N1-infected chicken immune cells. We will explore how a viral protein, NS1, modulates the activities of MAP kinases through interaction with a host protein NF90. Our findings from this proposal will further our understanding at a molecular level on high pathogenicity and pathogenesis of HPAI infection in birds.

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APPROACH: 1. Examine the activation of MAPK in H5N1 AIV-infected chicken macrophages: Chicken macrophages HTC will be used in the study. 3 ml cells (1x10>6 cells/ml) will be plated on 3 cm plates in the RPMI growth medium and used for infection. The virus inoculation and sample preparation will be carried out at the BSL-3 facility, Medical Biosciences Building, UMN. An H5N1 isolate, A/Vietnam/1203/2004, which is highly pathogenic in chickens, and its attenuated form with the deletion of the basic amino acid stretch in the HA gene, will be used. Cell lysates or total RNA will be prepared at various time points and used for SDS-PAGE and western blot analyses. Chicken specific anti-ERK1, p38 and JNK and anti-phospho-ERK, phospho-p38 and phospho-JNK antibodies will be used to determine the levels of the MAPK expression and the temporal activation status. 2. Comparing proinflammatory cytokine responses in HPAI and LPAI-infected chicken macrophages Total RNA will be reverse transcribed (RT), and cDNA will be used for real-time PCR to determine the transcriptional levels of selected chicken cytokines and chemokines, which will include IL-1â, IL-6, IL-8, TNF-a. RANTES (2, 3). SYBR Green-based RT-PCR will be carried out and Ct for each analyzed gene will be normalized against GAPDH (3). To determine how the cytokine responses are regulated by MAPK, we will use the specific inhibitors to suppress the individual MAPK pathway in infected chicken macrophages. Chicken macrophages will be pre-treated with MAPK inhibitors, U0126 (ERK), SB 203580 (p38), and InSolution JNK Inhibitor II (all from CalBiochem) at their optimal concentration. Total RNA will be prepared from the treated and untreated cells at various time points for realtime RT-PCR analyses, to measure the changes of proinflammatory cytokines/chemokines, to determine how cytokine responses to the H5N1 infection are regulated by individual MAPK. 3. Elucidate the role of NF90 in the regulation of MAPK activation through interacting with viral NS1: We will examine: A) how the NS1 interacts with NF90 at molecular level; and B) if the interaction affects the activation of MAPK. We will generate a series of H5N1 NS1 deletion mutants, which will be cloned into pRK5-flag vector. Co-immunoprecipitation (Co-IP) and western blot analyses will be performed to narrow down the essential and minimal fragment in NS1 which binds to NF90. We intend to define the critical amino acid residues, which are responsible for the interaction between NS1 and NF90. Mutant NS1 cDNAs will be generated with site-directed mutagenesis, in which these critical amino acids will be substituted; recombinant viruses with mutant NS1 will be generated in 293T/MDCK cultures. Using the mutant H5N1 viruses, deficient of interacting with NF90, to infect HTC cells, we plan to investigate if the phosphorylation and activation of MAPK are decreased in mutant H5N1-infected cells, because of increased MKP-1, using the approach as mentioned above. The cells are infected with the H5N1 viruses which contains the NS1 protein, deficient of interacting with and thus, sequestering NF90.