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Infection of chickens with highly pathogenic (HP) avian influenza (AI) (HPAI) of the H5N1 Asian lineage has become a major problem for the poultry industry causing acute to peracute mortality in chickens and to a lesser degree in ducks. Thus far, there is little information available on the mechanisms that are the cause of the acute to peracute death. Transmission of this virus to humans may occur sporadically but if it occurs is lethal in a high percentage. Virus isolated from the human lethal cases has frequently lysine (K) at position PB2-627, while the presumptive original poultry virus has a glutamic acid (E) at that position. We have shown that infection of chickens with reverse genetics-generated A/Vietnam/1203/04 strain (VN/1203) and the isogenic strain K>E, in which K has been replaced by E, results in peracute mortality within 48 hours post infection (PI) without showing lesions. Virus replication was detected at 24 but not at 12 hours PI with a suggestion of higher titers for VN/1203 than for K>E. Infection of chickens also resulted in activation of several proinflammatory cytokines as early as 24 hrs PI with significant differences between the two isogenic strains. Both viruses caused approximately 30% mortality in ducks between 6 and 10 days PI.
<P>We have the following specific objectives for the proposed research: <Ol> <LI> To determine if PB2-627 influences the pathogenesis directly we will infect chickens with the low pathogenic (LP) isogenic VN/1203 and K>E viruses. The use of the LP isogenic viruses is expected to be useful in determining if K at PB2-627 can enhance systemic cytokine changes in chickens leading to pathology in the absence of the peracute mortality. The latter is associated with infection with the HP strains and the peracute mortality may prevent the expression of pathological differences between the two strains. We expect that the results of these studies will provide information on the importance of these changes for the pathogenesis and pathogenicity. <LI>To further examine the development of hypercytokinemia in chickens and ducks in relation to virus replication we will infect chickens and ducks with the HP isogenic pair of viruses and determine at specific time points virus titers, cytokine changes and determine if thrombocytes play a role in the pathogenesis. Thrombocytes are an important source of cytokines in chickens. <LI>To determine if thrombocytes play a role we will also conduct in vitro experiments by infecting chicken and duck thrombocytes with the HP and LP isogenic viruses and measure release of IL-6 by stimulation of mouse TDL cells and mRNA for TLR3, IL-6, IL-13, IFN-alpha and -beta, and viral nucleoprotein by qRT-PCR.<LI> To determine if the type of amino acid at NS1-149 plays a role in modulating the production of IFN-alpha and beta in chickens and ducks infected with the isogenic pair of viruses. Both VN/1203 and K>E increase the production of these interferons within 24 hrs PI in chickens, but the importance of the amino acid (G) at NS-149 in this isogenic pair of strains has not been examined.
NON-TECHNICAL SUMMARY: The current increase in outbreaks of highly pathogenic avian influenza caused by Asian H5N1 strains has been well publicized. These outbreaks are a concern for the poultry industry and for the risk of developing a pandemic. The reasons for the high mortality are not fully elucidated. Avian influenza viruses can rapidly mutate and as a consequence isolated viruses may have a large number of variations in expressed proteins. To examine the importance of individual changes virus is cloned using reverse genetics, which then allows the introduction of specific mutations. We are examining the importance of a specific mutation resulting in an amino acid change from glutamic acid to lysine at position 627 of the polymerase protein PB2. This change has been identified as a potential pathogenicity factor in human isolates. In our preliminary studies in chickens and ducks we used the reverse genetics generated A/Vietnam/1203/04, which has lysine at PB2-627, and the isogenic strain K>E, in which lysine has been replaced by glutamic acid. The results showed that the former caused significant higher levels of cytokines than the latter. The purpose of this project is to further examine the importance of this change for the pathogenesis of infection in chickens and ducks. Specifically we plan to examine the role of thrombocytes in virus dissemination and cytokine release, the importance of the cytokines when birds are infected with these isogenic viruses which the have been mutated to the low pathogenic form and the potential interactions between PB2-627 and the NS1 gene.
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APPROACH: The basic approach for each animal experiment is similar in design, but the time points for collection of samples will vary depending on a) the type of virus strains (e,g, VN/1203 and K>E, these strains with the HA cleavage site mutated to the LP variant, and VN1203 and K>E with mutations at NS1-149), b) if chickens or ducks are used, and c) the specific question to be addressed. In general, four-week-old chickens or ducks will be infected by eye/nose drop and the following samples will be obtained for virus isolation and cytokine analysis: spleen, lung and brain. Virus titers will be determined in VERO cells and cytokine mRNA levels will be measured by real time quantitative (q)RT-PCR. In first instance we will concentrate on measuring IFN-alpha and -beta, TLR3, IL-6 and IL-13, but other cytokines may be included. Tissues will also be processed for histopathology and immunohistochemistry using standard techniques. Selected samples will be analyzed by microrarrays to determine global differences in host gene regulation. Criteria for inclusion will be based on the outcomes of all other assays using the samples showing the greatest differences between isogenic pairs of virus strains. If major differences are found we will confirm the differences by qRT-PCR. We will examine the potential role of thrombocytes in the cytokine storm associated with HPAI in two ways. As part of all animal experiments we will obtain blood samples for thrombocyte purification and isolation. These cells will be used for virus isolation and qRT-PCR. We will compare duck and chicken thrombocytes for susceptibility to virus infection virus-induced cytokine release. These cells will be cultured and exposed to live and inactivated VN/1203 and K>E. Samples will be obtained immediately after infection, at 6 and 12 hours and analyzed for IL-6 and PGE2 release, and virus replication. IL-6 and PGE2 are selected because we can use the 7-TD1 mouse cell line to measure biological activity of IL-6 and PGE2.