Implications of collective dissemination for segmented viruses Restricted; Files Only
Sethi, Meher (Fall 2025)
Abstract
Collective viral dissemination refers to the transmission of multiple virions as consolidated infectious units. This mode of spread plays an important role in shaping infection dynamics, promoting genetic diversification, and influencing viral evolution. In this thesis, I investigate how virion aggregation, a specific form of collective dissemination, modulates early infection parameters. These parameters include the number of virions delivered to individual cells, the scale of productive infection as assessed by the percentage of infected cells and overall viral yield, and the extent of genetic diversity generated during the earliest rounds of viral replication.
Using mammalian orthoreovirus (reovirus) as a genetically tractable model system, I characterize how environmental pH regulates the reversible organization of virions into aggregated or dispersed states. I then examine how these pH-dependent organizational states influence early infection outcomes. Acidic conditions promote the formation of multi-virion aggregates, whereas basic pH disperses them into individual particles. These environmentally driven shifts in virion aggregation enhance multi-virion entry events, increase co-infection frequency at low multiplicities of infection, and broaden the diversity of progeny that emerges during the initial phases of multi-cycle infection. These effects occur even when only a small number of cells are initially infected, indicating that collective dissemination can rapidly generate early population diversity with the potential to influence downstream evolutionary trajectories.
I further demonstrate that saliva, a host-associated extracellular environment relevant to natural transmission, induces aggregation of influenza A virions. This aggregation can potentially modulate infectivity and alter the composition of early viral output, highlighting the broader relevance of environmentally mediated collective dissemination across viral systems.
Finally, I consider how the principles identified in this work may extend to nontraditional environments, including confined or extreme settings such as spacecraft and the International Space Station. In such settings, environmental conditions and close-contact transmission may amplify the evolutionary impact of collective viral dissemination.
Overall, this thesis establishes collective viral dissemination as a fundamental mechanism influencing infection success, early diversification, and evolutionary potential. The findings presented here provide a conceptual and mechanistic framework for understanding how collective viral interactions can contribute to viral persistence, adaptation, and transmission across diverse biological and environmental contexts.
Table of Contents
Chapter 1: Introduction…………………………………………………………………...12 - 69
A. Collective dissemination…………………………………………………………….12 - 25
· Introduction………………………………………………………………………….12
· Mechanisms of collective dissemination…………………………………………….12
· Implications for viral infectivity and genetic diversity……………………………....15
· Evolutionary and fitness implications………………………………………………..15
· Functional benefits of collective dissemination……………………………………...16
· References………………………………………………………………………17 - 25
B. Mammalian orthoreovirus…………………………………………………………..26 - 53
· Discovery…………………………………………………………………………….26
· Mammalian orthoreovirus serotypes……………………………………………..…..26
· Broad host-range and zoonotic potential…………………………………………….27
· Pathogenesis and human infections……………………………………………….....27
· Environmental surveillance of reoviruses…………………………………………....29
· Structure……………………………………………………………………….……..29
· Replication cycle……………………………………………………………………..31
· Reoviruses and reassortment…………………………………………………..……..34
· Reoviruses and the gut pH environment……………………………………………..35
· Figures and Tables………………………………………………………………37 - 40
o Figure 1: Cryo-electron microscopy (cryo-EM) imaging of T3D reovirus particles…………………………………………………………………………….…37
o Figure 2: pH gradients across the human gastrointestinal tract………….…..38
o Table 1: List of dsRNA segments of mammalian orthoreovirus type (T3D) and their functions………………………………………………………………………..39
o Table 2: Cellular receptors and attachment factors for mammalian orthoreovirus…………………………………………………………………………40
· References………………………………………………………………………41 - 53
C. Influenza viruses……………………………………………………………….…....54 - 69
· Introduction…….…………………………………………………………………….54
· Epidemiology………………………………………………………………………...55
· Host range and cross-species transmission of IAV…………………………………..56
· IAV structure………………………………………………………………………....57
· IAV life cycle…………………………………………………………………….…..59
· Table…………………………………………………………………………….……62
o Table 3: List of genome segments of influenza A virus and their functions….62
· References…………………………………………………………………..…..63 - 69
Chapter 2: Virion aggregation shapes infection dynamics and evolutionary potential………………………………………………………………………………..….70 - 117
A. Abstract…….…………………………………………………………………………….70
B. Importance……………………………………………………………………………….71
C. Introduction…………………………………………………………………………71 - 73
D. Results………………………………………………………………………………74 - 91
· Reovirus aggregation is pH dependent, reversible and influenced by buffer composition………………………………………………………………………….74
o Figure 1: Reovirus aggregation is enhanced near the isoelectric point and further tuned by ionic properties of the buffer..…… …………………..……75
o Supplementary Figure 1: Low pH buffers and PBS are cytotoxic to L929 cells, and reovirus aggregation is reversible and sensitive to buffer pH.……….…78
o Supplementary Figure 2: Optimization of aggregation conditions to reduce cellular cytotoxicity.……… …………………………………………………79
o Supplementary Figure 3: Reovirus is stable in low pH buffers; buffer induced viral aggregation is reversible and pH dependent.…… ……………….……80
· Aggregation ensures coordinated delivery of viruses to cells……………………..…81
o Figure 2: Collective entry of reovirus aggregates into cells.…………………82
o Figure 3: Aggregation promotes multi-virion delivery to individual cells……83
o Figure 4: Aggregation increases the number of virions per infected cell…….84
· Aggregation promotes co-infection and genetic exchange………………………...…85
o Supplementary Figure 4: Validation of 1:1 WT-Var mixture and PBS as a negative control for co-infection assays.……… ………………………….…87
o Figure 5: Aggregation promotes co-infection and enhances reassortment…………………………………………………………..……...88
· Aggregation modulates infection dynamics………………………………………….89
o Figure 6: Aggregation reduces the number of infectious units.………………90
o Figure 7: Aggregation limits viral spread and reduces viral output.…………91
C. Discussion………………………..……………………………………………….…92 - 96
· Figure 8: Intercellular virus-virus interactions shape infection dynamics and influence viral evolution, with aggregation limiting spread while enhancing genetic diversity.…… …………………………………………………………………..……96
D. Material and Methods………………………………………………………...……97 - 106
E. Acknowledgments…………………………………………………………………..…..106
F. References…………………………………………………………………...……107 - 117
Chapter 3: Influenza A aggregation in human saliva…………………………………118 - 128
A. Introduction……………………………………………………………………………..118
B. Methods……………………………………………………………………..……120 - 121
C. Results……………………………………………………………………………122 - 123
· Influenza A virus aggregation is promoted in human saliva………………………..122
· Influenza A virus aggregation varies with saliva donor………………………….…122
D. Discussion………………………………………………………………………...123 - 124
E. Figures……………………………………………………………………………125 - 126
o Figure 1: Influenza A aggregates in saliva.………………………………...125
o Figure 2: Patterns of influenza A aggregation vary among saliva samples collected from different donors.……………… ……………………………126
F. References…………………………………………………………………..……127 - 128
Chapter 4: Viral evolution in space: implications of extraterrestrial conditions modulating evolution and transmission of viruses………………………………………………..129 - 189
A. Abstract…………………………………………………………………………129 - 130
B. Introduction……………………………………………………………………..130 - 133
C. Viral evolution………………………………………………………………..…133 - 139
o Mechanisms of viral evolution………………………………………………133 - 139
o Processes generating genetic variation………………………..……133 - 136
- Mutation…………………………………………………….….134
- Genetic exchange………………………………………………135
o Drivers of viral evolution……………………………………………136 - 138
- Migration……………………………………………………..…136
- Natural selection………………………………………..………137
- Genetic drift………………………………………………….…137
o Viral ecotypes across terrestrial and extraterrestrial environments…………..138 - 139
D. Extraterrestrial environments shape viral transmission dynamics…………….…139 - 144
o Within-host viral dynamics under spaceflight conditions…………………………140
o Between-host viral transmission in extreme environments……………………..…141
o Collective dissemination of viruses in space………………………………………142
E. Case study: Spacewalks and viral evolution……………………………………...144 - 146
F. Discussion…………………………………………………………………...……146 - 149
G. Acknowledgments………………………………………………………………………149
H. References………………………………………………………………………..149 - 178
I. Figures and Tables………………………………………………………………..179 - 189
o Figure 1: Forces governing viral evolution………………………..179 - 180
o Figure 2: Potential fitness benefits conferred by collective viral dispersal………………………………………………….…………………...181
o Table 1: Selected spaceflight stressors and their effects on virus-host dynamic…………………………………………………………….182 - 186
o Table 2: Modes of viral transmission in space habitats……………187 - 189
Chapter 5: Discussion…………………………………………………………………...190 - 203
· pH as a potential regulator of virion aggregation in the gut………………………..190
· Potential implications of ISVP formation on this study……………………………192
· Potential cost of collective dispersal: Phenotypic hiding…………………………..192
· Collective dissemination as a potential driver for viral adaptation…………………194
· Implications for spaceflight and broader evolutionary contexts……………………195
· Conclusions…………………………………………………………………………196
· Figure……………………………………………………………………………….198
· References……………………………………………………………………..199-203
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