Influenza A virus (IAV) circulates in waterfowl, causing mostly asymptomatic infections. IAV can undergo host adaptation and evolve to cause significant disease and mortality in domestic poultry and mammals, applying an enormous socio-economic burden on society. Sporadically, IAV causes global pandemics in man due to its zoonotic nature, and this can result in millions of deaths worldwide during a single outbreak. Host adaptation of IAV is an incompletely understood phenomenon, but is known to involve both host and viral determinants. It is essential to improve the understanding of the factors governing host range and pathogenicity of avian IAV, especially given the absence of a universal influenza vaccine and a limited weaponry of effective antiviral compounds. This study set out to improve the understanding of host adaptation of avian IAV, focussing on segment 8 (NS segment) of the virus genome. The NS segment of non-chiropteran IAV circulates as two phylogenetically distinct clades – the ‘A-’ and ‘B-alleles’. The A-allele is found in avian and mammalian viruses, but the B-allele is considered to be almost exclusively avian. This might result from one or both of the major NS gene products (NS1 and NEP) being non-functional in mammalian host cells, or from an inability of segment 8 RNA to package into mammalian-adapted strains. To investigate this, the NS segments from a panel of avian A- and B-allele strains were introduced into human H1N1 and H3N2 viruses by reverse genetics. A- and B-allele reassortant viruses replicated equally well in a variety of mammalian cell types in vitro. Surprisingly, the consensus B-allele segment 8 out-competed an A-allele counterpart when reassortant H1N1 viruses were co-infected, with the parental WT segment 8 being most fit in this system. A- and B-allele NS1 proteins were equally efficient at blocking the mammalian IFN response both in the context of viral infection and in transfection-based reporter assays. Consensus A- and B-allele H1N1 viruses also caused disease in mice and replicated to high virus titre in the lung. Interestingly, the B-allele virus induced more weight-loss than the A-allele, although the parental WT virus was most pathogenic in vivo. To re-address the hypothesis that B-allele NS genes really are avian-restricted, the relative rates of independent Aves to Mammalia incursion events of A- and B-allele lineage IAV strains was estimated and compared using phylogenetic analyses of all publically available segment 8 sequences. 32 A-allele introduction events were estimated compared to 6 B-allele incursions, however the total number of avian Aallele sequences outnumbered B-allele sequences by over 3.5 to 1, and the relative rates of introduction were not significantly different across the two lineages suggesting no bias against avian B-allele NS segments entering mammalian hosts in nature. Therefore, this study provides evidence that avian B-allele NS genes are not attenuating in mammalian hosts and are able to cause severe disease. Thus, this lineage of IAV genes, previously assumed to be avian-restricted, should be considered when assessing zoonotic potential and pandemic risk of circulating avian IAVs.

Doctor of Philosophy / Department of Biology / Stefan Rothenburg / Vaccinia virus is the prototypic poxvirus that has been widely used as a model for investigating poxvirus biology and genetics. Like several members of the Poxviridae family, vaccinia virus can infect several different species including mice, cows and humans. Because the entry of poxviruses into a host cell relies on ubiquitously expressed surface molecules, which are found in many species, the ability of poxviruses to infect and replicate in different host cells primarily depends on their ability to subvert the host’s innate immune response. One critical barrier to infection is overcoming the general shutdown of protein translation initiated by the cellular protein kinase PKR. PKR detects cytoplasmic double-stranded (ds) RNA generated during infection by the replicating virus, which activates it to phosphorylate the alpha-subunit of the eukaryotic translation initiation factor 2 (eIF2) and suppress general translation. Poxviruses are large viruses with dsDNA genomes that encode around 200 genes. Several of these genes are known as host range genes and are important for replication in different host species and many interact with components of the host immune response to promote viral replication. Two genes in vaccinia virus, called E3L and K3L, are known inhibitors of PKR and have previously been shown to be important for virus replication in cells from different species. The molecular explanation behind their host range function, however, is unknown. The main goal of the research presented in this thesis is to determine the molecular mechanisms for the host range function of vaccinia virus E3L and K3L, particularly in different hamster host cells. Along with an analysis of vaccinia virus host range genes, we have used genome-wide comparisons between host-restricted poxviruses in the Leporipoxvirus genus to parse out the potential genomic determinants of host range restriction in this clade of poxviruses. The overarching aim of this thesis work is to better understand the molecular mechanisms for host range in poxviruses.