Effects of Packaging Signal Divergence on Influenza A Virus Reassortment Restricted; Files Only

White, Maria (Fall 2019)

Permanent URL: https://etd.library.emory.edu/concern/etds/qn59q5063?locale=es
Published

Abstract

Influenza A virus (IAV) is an RNA virus with a segmented genome. Since the IAV genome comprises eight different segments, IAVs can exchange genetic material in a process called reassortment. Reassortment occurs when two different IAVs co-infect the same cell and mixing of the two viral genomes takes place within the cell. Reassortment therefore allows the production of new viruses that are different from both parental viruses. Regions of the IAV genome called packaging signals serve to direct incorporation of the eight segments during virus assembly, and these packaging signals are found on both termini of each of the eight IAV segments. Since these packaging signals are specific to each segment and to each strain of IAV, we hypothesized that nucleotide differences within the packaging signal regions could limit reassortment efficiency. To test this hypothesis, we used two viruses representative of the two seasonal IAV lineages currently circulating in the human population (H3N2 and H1N1), and made virus pairs that encoded the same proteins but different packaging signals on a single segment. We then evaluated how frequently segments carrying matched vs mismatched packaging signals were incorporated into reassortant progeny. We found that matched packaging signals were preferred on the HA segment, but not on the NA or NS segments. These data suggest that the movement of NA and NS segments between seasonal IAVs is unlikely to be restricted at the level of packaging, while movement of the HA segment would be more constrained. Therefore, our results indicate that the importance of packaging signal differences in IAV reassortment is segment dependent. Furthermore, our data suggest that the packaging signals of the HA segment could be an important factor in determining the likelihood that two IAV strains of public health interest will undergo reassortment.

Not only can reassortment increase the diversity of viruses circulating within a given host species, but it can also facilitate transfer of novel IAVs to a new host species. IAV pandemic strains are formed in part by the introduction of an HA segment from a nonhuman virus, such as an avian virus, into a human virus. Since the mixing of avian and human IAVs can result in the formation of a pandemic strain, we next addressed how likely it would be for an HA segment from an avian IAV to reassort with a human IAV. We tested the compatibility of HA packaging signals from two avian viruses that pose a threat to human health (H5N8 and H7N9) with a human seasonal H3N2 strain. We showed that sequence differences in the packaging signals between these avian and human IAV HA segments limited the potential for reassortment to occur. We did find, however, that human viruses still incorporated the avian virus HA segments at a low level in animals that were co-infected with both IAVs. This observed low level of avian virus HA segment incorporation into human viruses could be significant if the reassortant virus had a fitness advantage in the human population, such as escape from pre-existing immunity. Overall, these findings offer important insight into the mechanisms that underlie the emergence of new IAVs and highlight the continued need for IAV surveillance, especially in areas where avian and human IAVs are in close contact.

Table of Contents

Chapter I: Introduction………………………………………………………………………1-53

    Figures……………………………………………………………………………………27-30

         Fig. 1……………………………………………………………………………………....27

         Fig. 2………………………………………………………………………………………28

         Fig. 3…………………………………………………………………………………...29-30

    References………………………………………………………………………………...31-53

Chapter II: Heterologous Packaging Signals on Segment 4, but not Segment 6 or Segment 8, Limit Influenza A Virus Reassortment…………………………………………………...54-100

    Abstract and Importance………………………………………………………………..55-56

    Introduction……………………………………………………………………………...56-59

    Materials and Methods…………………………………………………………………..59-65

    Results…………………………………………………………………………………….65-72

    Discussion………………………………………………………………………………...72-77

    Figures……………………………………………………………………………………78-86

         Fig. 1………………………………………………………………………………………78

         Fig. 2………………………………………………………………………………………79

         Fig. 3………………………………………………………………………………………80

         Fig. 4………………………………………………………………………………………81

         Fig. 5………………………………………………………………………………………82

         Fig. 6…………………………………………………………………………………...83-84

         Fig. 7…………………………………………………………………………………...85-86

    Tables……………………………………………………………………………………..87-89

         Table 1……………………………………………………………………………………..87

         Table 2……………………………………………………………………………………..88

         Table 3……………………………………………………………………………………..89

    References……………………………………………………………………………….90-100

Chapter III: Serial Passage of an Influenza A Virus Containing Heterologous Packaging Signals on Segment 4……………………………………………………………………...101-127

    Abstract………………………………………………………………………………….…102

    Introduction…………………………………………………………………………...103-104

    Results…………………………………………………………………………………104-108

    Discussion……………………………………………………………………………...108-112

    Materials and Methods……………………………………………………………….112-116

    Figures…………………………………………………………………………………117-120

         Fig. 1……………………………………………………………………………………..117

         Fig. 2……………………………………………………………………………………..118

         Fig. 3……………………………………………………………………………………..119

         Fig. 4……………………………………………………………………………………..120

    Tables…………………………………………………………………………………..121-122

         Table 1……………………………………………………………………………………121

         Table 2……………………………………………………………………………………122

    References…………………………………………………………………………...…123-127

Chapter IV: H5N8 and H7N9 Packaging Signals Constrain HA Reassortment with a Seasonal H3N2 Influenza A Virus………………………………………………………..128-171

    Abstract and Significance……………………………………………………………..129-130

    Introduction…………………………………………………………………………...130-132

    Results………………………………………………………………………………….132-137

    Discussion……………………………………………………………………………...137-141

    Materials and Methods………………………………………………………………..141-144

    Figures…………………………………………………………………………………145-164

         Fig. 1………………………………………………………………………………...145-146

         Fig. 2……………………………………………………………………………………..147

         Fig. 3………………………………………………………………………………...148-149

         Fig. 4……………………………………………………………………………………..150

         Fig. 5………………………………………………………………………………...151-152

         Fig. S1……………………………………………………………………………….153-154

         Fig. S2……………………………………………………………………………….155-156

         Fig. S3……………………………………………………………………………….157-158

         Fig. S4……………………………………………………………………………………159

         Fig. S5……………………………………………………………………………………160

         Fig. S6……………………………………………………………………………….161-162

         Fig. S7……………………………………………………………………………….163-164

    Tables…………………………………………………………………………………..165-166

         Table S1………………………………………………………………………………….165

         Table S2………………………………………………………………………………….166

    References……………………………………………………………………………...167-171

Chapter V: Discussion……………………………………………………………………172-181

    References……………………………………………………………………………...179-181

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