Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology

First Advisor

Turner, Paul

Abstract

Dengue virus is the world’s preeminent arthropod-borne virus that infects hundreds of millions of people each year, and circulates in four distinct serotypes named 1, 2, 3, and 4. Dengue virus cycles between mosquito and human hosts in a transmission cycle called the endemic cycle, which is responsible for the majority of recorded dengue infections worldwide. However, dengue also exists in a second transmission cycle called the sylvatic cycle, which mirrors the endemic cycle with wildlife, including non-human primates, replacing humans in this transmission cycle. Phylogenetic analyses support the hypothesis that spillover events from the sylvatic cycle into human populations hundreds of years ago seeded what is the now the endemic cycle of all four dengue virus serotypes. Few sylvatic strains have been isolated, and these strains have been chronically understudied within dengue research. Additionally, the characterization of these strains has largely occurred with sylvatic dengue-2 strains. Only a handful of studies have examined the ability of these strains to replicate on a variety of relevant hosts from both the sylvatic and endemic cycles. Also, it is important to understand how sylvatic and endemic dengue strains evolve over time and adapt to environmental challenges to see if these adaptations are similar or distinct. However, to date, only one study conducted experimental evolution with both sylvatic and endemic dengue strains and measured the ability of these evolved strains to replicate on a novel host. In this thesis, we looked to deepen our characterization of sylvatic dengue-4 strains and understand how they evolve and compare these phenotypes and genotypes to comparable endemic dengue-4 strains.

In the first chapter, we characterized three sylvatic and two endemic strains via replication kinetics on a variety of mosquito and monkey cell lines that represent plausible hosts for either the sylvatic or endemic cycle or both. Before initiating these assays, we also confirmed via genetic analyses that our viral stocks did not undergo an excessive amount of passaging in tissue culture that could have resulted in a higher-than-expected number of single nucleotide polymorphisms to accumulate in the genome and therefore be poor representations of sylvatic and endemic strains. The cell types we included in these analyses were three mosquito cells lines (C6/36, U4.4, and AAG2) and a monkey cell line (Vero). Using a probe-based qRT-PCR assay, we observed similar dynamics across all virus strains but differences among cell lines, with higher viral copy numbers on C6/36 and Vero cells at day five post-infection (pi). We hypothesize that the lower viral copy number on U4.4 and AAG2 cells is attributable to their intact RNAi system, which is an important anti-viral response of mosquito cells that C6/36 cells lack. When stratifying our data by cell type rather than strain, our data does not show statistically significant differences in the replication kinetics between endemic and sylvatic strains for C6/36 and Vero cells, which corroborates previously published data, and also U4.4 cells, which is a novel finding. We also performed an RNA sequencing (RNA-seq) analysis on a subset of strains (one endemic and one sylvatic) to understand what impact dengue infection has on the transcriptome of Vero cells, a relevant monkey cell line. The cells infected by the endemic strain experienced a greater number of genes statistically up-and-downregulated when compared to those infected by the sylvatic strain. The pathway analysis software Shiny GO showed that the upregulated genes from endemic and sylvatic infection were found in pathways related to anti-viral replication and host defense. However, the sylvatic strain also experienced an upregulation of the host pathways that assist in viral replication. The fact that fewer genes were up and down regulated by the presence of sylvatic dengue coupled with the viral replication pathway being upregulated potentially indicates to a more tolerance phenotype of sylvatic dengue when compared to endemic dengue, but it important to stress that these data only came from a single sylvatic and endemic strain of dengue virus.

In the second chapter, we used experimental evolution to explore how three sylvatic strains and two endemic strains of dengue virus serotype 4 are challenged to adapt and evolve in a variety of environments, which to our knowledge is the first study conducting experimental evolution with several sylvatic strains of any serotype, and also the first experimental evolution study to include sylvatic dengue-4. Our experiment consisted of twelve in vitro serial passages in three replicated treatment environments: Vero cells alone, U4.4 cells alone, and alternating passages of Vero and U4.4 cells. In addition, we examined a “Combo” of viruses, where a single pair of sylvatic and endemic viruses were challenged to adapt in these same three environments. To determine the titer of our populations, and therefore the change in carrying capacity, we carried out a focus forming assay since sylvatic dengue does not plaque in a standard plaque assay. Additionally, we conducted whole genome sequencing (WGS) at the endpoint of the evolution experiments, so we could detect single nucleotide polymorphisms (SNPs), which may help explain the phenotypic results we observed. The Vero only populations saw minimal increases in carrying capacity with some replicates actually decreasing in carrying capacity, and this pattern was observed across all strains. This treatment also had the most SNPs detected at the endpoint, and while some localized to sequences that encode for proteins that mediate host entry (envelope) and antagonize the host’s immune system (NS4B, NS5), among other functions, there was no clear pattern of localization, and these SNPs did not appear across biological replicates. The U4.4 only treatment saw the starkest outcome of all experiments with the two endemic strains and the Combo increasing their carrying capacity, while all sylvatic strains went extinct. One endemic strain, P9-592, saw several identical SNPs arise across biological replicates, but these did not occur in genes we predicted to be sites of SNPs. Finally, the Alternating treatment saw an increase in carrying capacity for all strains which was uniform across strains when measured on U4.4 cells and varied when measured on Vero cells. This treatment saw the fewest SNPs arise, but the majority were found across biological replicates, even though there was no consistent pattern in the genes to which they localized. Additionally, reads from our WGS of our Combo populations mapped to the env gene of both strains, which could imply that both strains persisted to the endpoint of all evolution experiments, even in the U4.4 only evolution experiment, where all sylvatic strains went extinct. This could imply that in some environments, co-infection with an endemic strain can rescue a sylvatic strain from extinction.

The third chapter explored the ability of evolved viral populations from Chapter 2 to replicate on a novel host, the human hepatocyte cell line HepG2, for one passage. Often during experimental evolution, a viral population adapts to its environment, which can then directly shape its performance on a novel host. Since the HepG2 cell line constitutively produces interferon, we wanted to explore the impact that evolving in the absence or presence of innate immunity would have on the ability of viral populations to emerge on this new host by utilizing a probe-based qRT-PCR assay. Vero only populations increased in carrying capacity across all strains except for the endemic strain P9-592. Even though these populations evolved in an environment lacking interferon, we hypothesize that the phylogenetic relatedness between a monkey and human meant that Vero cell line adaptations of our viral populations potentially were also beneficial in the HepG2 environment. Interestingly, U4.4 only populations saw divergent outcomes, with endemic strain P7-1006 increasing in carrying capacity, while P9-592 decreased in carrying capacity. We suggest that these strains adapted to the RNAi positive environment of U4.4 cells in different ways, with P9-592 having accumulated more SNPs by the endpoint of the evolution experiment for example, which potentially led to their respective outcomes in carrying capacity. Finally, the Alternating populations decreased in carrying capacity across all strains with many replicates falling below the limit of detection of our assay, implying extinction. We hypothesize that the host cycling nature of the Alternating evolution experiment allowed for increase in capacity on Vero and U4.4 cells, but this may have reduced genetic diversity, which in turn did not allow for these populations to successfully increase their carrying capacity in a novel host. Sequencing data was only of sufficient depth for our Vero only strains and the U4.4 only P7-1006 strain due to the low viral copy number of many of our populations. For the populations for which we did have sufficient sequencing data, only four SNPs were observed across all strains and replicates, which suggests that the phenotypes we observed during the novel host assay were potentially not due to new SNPs that arose during a single passage on the novel host.

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