The Avian Innate Immune Response to AIV Infection and the Effect of the Viral Non-structural Gene 1 on Host Cell and Viral Function PDF Download

Author: Sean Cody Adams
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ISBN: 9781303791550
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Languages : en
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Book Description
The Influenza A Virus (IAV) employs a variety of strategies in order to disarm the host immune response, most of which are carried out by the non-structural 1 protein encoded in the eighth viral genome segment. One such strategy is to blunt the innate immune response by preventing the expression of the type I interferons (IFN), other pro-inflammatory cytokines and immune-related genes. Disruption of the interferon-signaling pathway is a central component of viral infection in both mammalian and avian hosts. However, there is considerable variability in virus capacity to disrupt this signaling pathway as well as variation between species of the host expression of immune-related genes in response to infection with the same virus. Moreover, the clinical outcomes of viral infection differ among avian species; ducks harbor persistent, asymptomatic infections, where chickens display more robust responses and present symptomatically with respiratory signs, decreased egg production, and mortality. Avian species are not well understood as hosts of IAV. The general roles of avian species in the transmission, perpetuation, and evolution of IAV is well known although detailed information is lacking including knowledge of avian immune responses and their regulation by IAV. As a consequence, IAV from birds are frequently adapted to and subsequently assessed in mammalian hosts such as mice and ferrets for immune function and viral transmission studies, the results of which are extrapolated to encompass the generic non-human response to avian influenza. To address this gap in knowledge regarding the avian response to IAV, the following chapters of this thesis describe the early responses to IAV infection on a molecular signaling level and compare them in cells derived from chickens and ducks. In Chapter 2, by infecting peripheral blood mononuclear cells (PBMC) from avian hosts that have differences in responses to IAV infection and play different roles in the natural history of IAV with the same low pathogenic avian influenza virus, we identified differences in the expressions of key immune-related genes. Interestingly, the cytopathic effect of the virus was evident in chicken cells, whereas duck cells displayed no evidence of infection. Furthermore, the in vitro cytokine and immune-gene expression profiles between the two host's differed. Specifically pro-inflammatory molecules IL-1[beta] and IL-6 were highly upregulated in chicken cells, whereas duck cells expressed baseline levels, and a similar pattern was observed for both IFN-[beta] and IFN-[gamma]. Taken together, these patterns suggest a molecular mechanism for observed clinical presentation. Low pro-inflammatory and IFN levels are consistent with an asymptomatic response to IAV infections in ducks, while the robust responses in chickens are more consistent with symptomatic infections. These findings provide valuable insight into the IFN expression patterns induced by IAV and differences between the responses of avian hosts. A canonical response to avian IAV infections has been reported in mammalian hosts, where the IAV NS1 protein, antagonizes the expression of type I IFN at multiple levels of the cellular pathway. It is less clear how NS1 interacts with avian cellular proteins, and whether viral suppression of IFN expression is similar in birds generally or if the mechanism and magnitude of expression differs between the major classes of avian hosts. To further complicate the issue, the NS1 gene exists as two allelic variants. One variant, the A allele, has been identified in viruses infecting all susceptible hosts, mammalian and avian. The other variant, the B allele, is almost exclusively isolated from viruses infecting avian hosts with few exceptions. The two alleles are only 69% identical at the amino acid level, and the sequence differences imply the two alleles may function differently. The second aim in this body of work was to characterize the in vitro innate immune gene (the type I IFNs and downstream cytokines) expression of chickens, turkeys, and ducks in response to infection with A allele or the B allele reassortant viruses. This study identified key differences and similarities between the two alleles. First, viruses bearing the A allele replicated to higher titers in the cells of chickens and turkeys (species which share a more recent ancestor), whereas viruses bearing the B allele replicated to higher titer in duck cells (species more distantly related to chickens and turkeys). Second, the B allele also suppressed the expression of IFN-[beta] more effectively than did the A allele at a single time point in chicken and turkey cells. The differences between the alleles ended there; both A and B allele NS1 proteins modulated the expression of the other type I IFN and their downstream effector proteins Mx and 2'-5' oligoadenylate synthetase to similar extents. Chimeric constructs of the NS1 gene were used to identify regions of the NS1 gene responsible for the differences in replication titers and the expression of IFN-[beta] in chicken cells between A and B alleles. This assay identified the nucleotides spanning positions 1-164 as responsible for the differences in viral replication, whereas nucleotide positions 164-541 were responsible for the differences in IFN-[beta] expression in chicken cells. Combined, these results form the first comprehensive study of IAV and NS1 alleles in avian species. The final aim of this study was to characterize the geographic distribution, prevalence, and selective pressure driving amino acid changes of the NS1 alleles in viruses isolated from North American chickens, turkeys, and ducks. In addition to online databases, sequences were obtained from the California H6N2 outbreak in chickens, which took place between 2000 and 2005. This study identified a higher than expected proportion of B alleles in circulation from 2002-2012. Interestingly, the H6N2 viruses were associated with a mixed population of viruses bearing either the A or the B allele, which is unusual since other outbreaks were associated with only one NS1 sequence. Lastly, a novel approach to identifying selective pressures was employed by examining the ratio of nonsynonymous to synonymous substitutions of specific functional regions of the NS1 gene and compared to the ratio of the entire gene. This approach identified that the carboxyl terminus of the NS1 gene is undergoing positive selective pressure which is driving variation in this region.