Influenza poses a significant global health risk due to the high morbidity and mortality associated with endemic strains and the pandemic potential of novel strains. This research characterizes the zoonotic risk of emerging influenza viruses from domestic animals. Chapter two investigates the genetic compatibility of pandemic H1N1 and emerging canine influenza H3N2 (CIV-H3N2), first identified in dogs in 2007 and currently responsible for outbreaks in shelters in the U.S. We generated 51 of the 127 possible reassortant viruses: 19 showed high-growth phenotypes and 13 replicated in mice lungs. A reassortant with the HA and NP genes derived from CIV-H3N2 transmitted efficiently by direct contact in ferrets and was more pathogenic than wild-type CIV-H3N2. Our results suggest that CIV-H3N2 reassortants may pose a moderate risk to public health and that the canine host should be monitored for emerging FLUAV. Chapter three is a seroepidemiological study of influenza A (FLUAV) exposure in shelter dogs in Mississippi, a population not previously investigated. We sampled 565 dogs from eighteen shelters statewide and identified 7/565 dogs (1.2%) ELISA positive (S/N < 0.70) for FLUAV exposure but no positive dogs by HI assay. Chapter 4 investigates the pathogenicity of emerging influenza D virus (FLUDV), first isolated from symptomatic pigs in 2011. Serology suggests feral swine are exposed to both FLUAV and FLUDV and we investigated whether coinfection with these two viruses enhances pathogenicity in domestic pigs. In single infections, 5/5 pigs shed FLUAV and 6/7 pigs shed FLUDV in nasal swabs; however, in the FLUAV+ FLUDV coinfection group 7/7 pigs shed FLUAV but only 1/7 shed FLUDV. FLUDV replicated better in the turbinate and soft palate of single infection group than the coinfection group, whereas FLUAV replicated equally well in both single and coinfection groups. Our data suggests coinfection is associated with less robust FLUDV replication in pigs. Collectively, this dissertation offers insight into emerging influenza viruses originating in dogs and pigs and potential competitive outcomes associated with coinfection.

Note:

Vaccination against the Influenza A virus (IAV) is often an important and critical task for much of the population, as IAV causes yearly epidemics, and can cause even deadlier pandemics. Designing the vaccine requires an understanding of the current major circulating strains of Influenza, as well as an understanding of how those strains could change over time to become either less harmful or more deadly, or simply die out completely. An error in the prediction process can lead to a non-immunized population at risk of epidemics, or even a pandemic. Presented here is a posterior predictive approach to generate emerging influenza strains based on a realistic genomic model that incorporates natural features of viral evolution such as selection and recombination. Also introduced is a sequence sampling scheme to relieve the computational burden of the posterior predictive analysis by clustering sequences based on their pairwise similarity. Finally, the impact of “evolutionary accidents” that take the form of bursts of evolution and or of recombination on the predictive power of our procedure is tested. An analysis of the impact of these bursts is carried out in a retrospective study that focuses on the unexpected emergence of a new H3N2 strain in the 2007-08 influenza season. Measuring the R2 values of both pairwise and patristic distances, the model reaches a predictive power of ∼40%, but is not able to simulate the emergence of the target Brisbane/10/2007 sequence with a high probability. The inclusion of “evolutionary accidents” improved the algorithm’s ability to predict HA sequences, but the prediction power of the NA gene remained low.

Vaccination against the Influenza A virus (IAV) is often an important and critical task for much of the population, as IAV causes yearly epidemics, and can cause even deadlier pandemics. Designing the vaccine requires an understanding of the current major circulating strains of Influenza, as well as an understanding of how those strains could change over time to become either less harmful or more deadly, or simply die out completely. An error in the prediction process can lead to a non-immunized population at risk of epidemics, or even a pandemic. Presented here is a posterior predictive approach to generate emerging influenza strains based on a realistic genomic model that incorporates natural features of viral evolution such as selection and recombination. Also introduced is a sequence sampling scheme to relieve the computational burden of the posterior predictive analysis by clustering sequences based on their pairwise similarity. Finally, the impact of “evolutionary accidents” that take the form of bursts of evolution and or of recombination on the predictive power of our procedure is tested. An analysis of the impact of these bursts is carried out in a retrospective study that focuses on the unexpected emergence of a new H3N2 strain in the 2007-08 influenza season. Measuring the R2 values of both pairwise and patristic distances, the model reaches a predictive power of ∼40%, but is not able to simulate the emergence of the target Brisbane/10/2007 sequence with a high probability. The inclusion of “evolutionary accidents” improved the algorithm’s ability to predict HA sequences, but the prediction power of the NA gene remained low.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 133-154). Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 133-154). Also available in print.

The influenza A viral hemagglutinin (HA) protein binds to sialic acid (SA) groups of cellular surface glycoproteins to achieve viral attachment and entry. The SA binding specificity of HA is one of the major determinants for controlling viral tropism and host specificity. Fibronectin (FN) is a ubiquitinious glycoprotein secreted on cell surface, either circulating in plasma, or as one of the best characterized components of the extra cellular matrix. With its binding properties towards different types of molecules and pathogens, it has been utilized by different bacterial and viral pathogens for binding, entry, propagation and pathogenesis. The binding affinity and region of plasma FN to influenza A viral glycoprotein was identified in early 1980s. Evidence also suggests the binding is SA associated. FN associates with different viral pathogens. However, evidence of FN direct involvement in influenza A pathogenesis remains unknown. The objective of this thesis is to test the involvement of cellular FN in influenza A viral infection. To perform the study, FN siRNA and anti-FN antibody were applied. This study demonstrated possible involvement of FN in the replication of human H1N1 and highly pathogenic avian H5N1 viruses. It also discovered that FN is very important for the replication of H1N1 virus, but not H5N1 virus. Interestingly, the result suggested that FN does not affect the initial virus-host binding, but it has an effect on post-attachment events. Key amino acid positions controlling the SA binding specificity of seasonal human or avian influenza A viruses have been identified in the HA. In this thesis, reverse genetics and mutagenic work identified that viruses with a α2,3-linked SA (SA α2,3) binding preference were not inhibited by anti-FN antibody, while viruses with a α2,6-linked SA (SA α2,6) specificity were severely inhibited. This surprising finding of SA binding preference related FN involvement in post-attachment event led to the further investigation on the structural involvement of FN and viral entry pathway analysis. The 9th and 10th of type III repeating units of FN form the cell-binding domain of the protein for cell attachment. From site specific antibody inhibitory studies, the cell binding region of FN near the synergy adhesion site(SAS) and Arg-Gly-Asp-Ser(RGDS) cell adhesion signal was identified to be important for the replication of viruses that have a α2,6 SA binding preference, but it was also found to be independent of α5β1 integrin receptor. After attaching to a host cell, the virus was internalized in an endosome via clathrin- or caveolin- mediated endocytosis. By application of pathway inhibitors, the FN association with viral entry pathway was evaluated. Though this study failed to identify a single specific FN mediated viral entry pathway, this pathway study indicated the possibility of FN various involvement in influenza viral entry. The study indeed indicated that viruses have difference SA binding preferences are different in their choices in viral entry pathways. This thesis did not only introduce cellular FN as a novel host factor, but also identified possible target and brought new light in the control of influenza A viral infection. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

Avian H5N1 viruses have perpetuated in poultry and caused sporadic human transmission since 1997. Vaccine candidates for the potential pandemic caused by H5N1 viruses have been continuously updated by World Health Organization. Multiple genetic lineages of H5N1 viruses which co-circulate and rapidly evolve in different regions, together with periodic population replacement of newly emerged genetic and antigenic variants in the field, pose great challenge for H5N1 vaccine candidate selection. The complexity of avian H5N1 viruses evolution raises an important issue for studying antigenic properties and also for projecting antigenic trend of this virus since the model established for the seasonal influenza viruses may not apply to H5N1 viruses which they are still in the animal phase. In contrast, the 2009 pandemic H1N1 viruses have established as another seasonal influenza viruses in humans. How will this swine originated viruses evolve genetically and antigenically in humans? For the first in human history, we are able to track the changes of pandemic viruses from the very beginning when they transmitted to human. This study focuses on antigenic and receptor binding properties of avian H5N1 viruses from 1997 to 2010 and 2009 pandemic H1N1 viruses from 2009 to 2011. It is found that avian H5N1 viruses continue to display highly diverse antigenic profile. The newly emerged H5N1 virus variants of clade 2.3.4 in 2008 and clade 2.3.2 in 2010 exhibit distinct antigenic properties as compared to the genetically similar viruses that were characterized previously. Receptor binding analysis showed H5N1 viruses still exhibit binding preference for avian type receptor. However, analysis of escape mutants selected from H5N1 viruses exposed to H5 monoclonal antibodies in cell based assay indicates that mutations in the conserved sites may cause switch of receptor binding specificity to human type or dual specificity for both human and avian. Based on antigenic and receptor binding analyses, it is found that the 2009 pandemic H1N1 viruses isolated from 2009 to 2011 are relatively stable. Most of the antigenic variants to monoclonal antibodies are transient and not able to become prevalent. It remains to be investigated if more significant antigenic variants may emerge in the coming seasons when population immunity prevails this virus. In conclusion, this study showed that clade 2.3 avian H5N1 viruses become increasingly antigenic distinct as compared to clade 2.1 and 2.2 viruses. Antigenic variation in antigenic sites may change receptor binding specificity in avian H5N1 viruses. The 2009 pandemic H1N1 viruses remain stable up to date but continue monitoring in coming seasons is necessary. / published_or_final_version / Microbiology / Master / Master of Philosophy

Vaccination against the Influenza A virus (IAV) is often an important and critical task for much of the population, as IAV causes yearly epidemics, and can cause even deadlier pandemics. Designing the vaccine requires an understanding of the current major circulating strains of Influenza, as well as an understanding of how those strains could change over time to become either less harmful or more deadly, or simply die out completely. An error in the prediction process can lead to a non-immunized population at risk of epidemics, or even a pandemic. Presented here is a posterior predictive approach to generate emerging influenza strains based on a realistic genomic model that incorporates natural features of viral evolution such as selection and recombination. Also introduced is a sequence sampling scheme to relieve the computational burden of the posterior predictive analysis by clustering sequences based on their pairwise similarity. Finally, the impact of “evolutionary accidents” that take the form of bursts of evolution and or of recombination on the predictive power of our procedure is tested. An analysis of the impact of these bursts is carried out in a retrospective study that focuses on the unexpected emergence of a new H3N2 strain in the 2007-08 influenza season. Measuring the R2 values of both pairwise and patristic distances, the model reaches a predictive power of ∼40%, but is not able to simulate the emergence of the target Brisbane/10/2007 sequence with a high probability. The inclusion of “evolutionary accidents” improved the algorithm’s ability to predict HA sequences, but the prediction power of the NA gene remained low.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.