Emerging viruses represent a significant threat to human health. Understanding the drivers of emergence, such as viral evolution, is a critical avenue to combat these pathogens. One specific group of emerging pathogens of interest is flaviviruses. Flaviviruses are arthropod-borne viruses (arbovirus) in the family Flaviviridae. The medically relevant flaviviruses can be divided into two groups – tick-borne and mosquito-borne. Included within the mosquito-borne flaviviruses group are dengue viruses 1-4 (DENV 1-4), which causes 400 million infections annually, and Zika virus (ZIKV), which caused over 128 million infections from 2013-2018. These viruses, which are cocirculating, share high sequence similarity in key antigenic regions. Because of these similarities, pre-existing immunity to DENV has been correlated with altered pathogenesis of subsequent ZIKV infections. Despite this, there has been little analysis of the effects of pre-existing DENV immunity on the evolution of subsequent flavivirus infection, despite being characterized for many other viruses. Given that mutation that could arise from cross-reactive immune selection could alter pathology or transmissibility, it is critical to assess the role of cross-reactive immune selection as an evolutionary driver. However, this line of research has historically been difficult due to the inherent toxicity of flavivirus infectious clones in bacteria. To mitigate the toxic nature of flavivirus clones, we developed several entirely in vitro workflows using a combination of rolling circle amplification (RCA) and replication cycle reaction (RCR). We demonstrated that RCA was a comparable substitute to traditional plasmid propagation using an alphavirus infection clone. We further demonstrated that RCR could be used to generate infectious clones by producing infectious clones of DENV2 and SARS-CoV-2, as well as demonstrating it could be used to introduce mutations into infectious clones by producing a D614G SARS-CoV-2 mutations. With this technology in place, we used in vitro directed evolution system, where we passaged ZIKV in convalescent patient serum to assess the role of cross-reactive immune selection as an evolutionary driver. After passaging, we performed next-generation sequencing to assess the impacts of cross-reactive immune selection on the viral populations and to identify mutations that arose post-passaging. We observed that ZIKV passaged in convalescent DENV serum had reduced diversity and divergence in the premembrane region. Within the convalescent DENV passaged population, we identified two mutations of interest with the dominant antibody binding region – E-V335I and NS1-T139A. These mutations were then introduced using our in vitro workflows. The resulting mutant viruses were then assessed for their replicative fitness in mammalian cell culture and mosquito models and their sensitivity to neutralization. We observed that while both E-V355I and NS1-T139A have increased fitness in mammalian cells, they had reduced fitness in mosquitoes. These results align with the trade-off hypothesis, which states that in a multi-host system, adaptation to one host reduces fitness in the other hosts. When we assessed the neutralization sensitivity of the mutants, we observed that while NS1-T193A was resistant to neutralization, E-V355I was more sensitive to neutralization. These results indicate that neutralization escape is not necessary for enhanced post-passaging in convalescent DENV serum. Our findings demonstrate that cross-reactive immune selection can generate several mutations with altered fitness in mammalian cells and mosquitos. This research is significant for both highlighting novel technologies to facilitate molecular virology and demonstrating that cross-reactive immune selection has the potential to alter the evolutionary trajectory of flaviviruses. This work provides critical information to understand how flaviviruses are evolving and emerging, and therefore critical information to address their threat to human health. / Doctor of Philosophy / Emerging viruses represent a significant threat to human health. We must understand what drives these viruses to adapt and evolve to respond to these threats. One virus family of extreme importance is the genera flavivirus. Flaviviruses are arthropod-borne viruses (arbovirus) that can be spread by the bites of ticks and mosquitoes. Included in the mosquito-borne flavivirus are dengue virus 1-4 (DENV1-4), which accounts for 400 million new infections annually, and Zika virus (ZIKV), which caused more than 128 million infection from 2013-2018. In addition to co-circulating, DENV 1-4 and ZIKV share several key similarities in their protein structures, which results in pre-existing DENV immunity effect how subsequent infections behave. The effect of pre-existing immunity on the evolution of these viruses has not been well established, despite similar studies being performed for other viruses. Given that the mutations that could arise from immune-driven evolution could alter disease severity or transmissibility, the impacts of immune-driven evolution must be characterized. However, the current tools available to perform this research are suboptimal, as the toxicity of flavivirus genomes hampers out ability to perform bacterial cloning, which has historically been necessary to develop and modify infectious clones. To mitigate the toxicity, we developed a "bacteria-free" workflow using emerging technologies like rolling circle amplification (RCA) and replication cycle reaction (RCR). With the technology in place, we propagated several generations of ZIKV or DENV in the presence of serum from human patients with a history of DENV infections. We then sequenced the viruses and identified mutations that arose during passaging. The mutations were then inserted using our bacteria-free workflow into infectious clones. The resulting viruses were assessed for their ability to replicate in mammalian cells, their ability to infect mosquitos, and their sensitivity to patient serum. We found that exposing ZIKV to serum from patients with pre-existing immunity to DENV can result in ZIKV developing several mutations. These mutations make the virus more effective at infecting mammalian cells and less effective at infecting mosquitos. This research is significant as it highlights novel technologies to aid researchers, and it demonstrates that pre-existing immunity has the potential to alter the evolutionary trajectory of flaviviruses. This information is critical in understanding flavivirus evolution and their emergence and therefore is critical to addressing their threat to human health.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/113850 |
| Date | 16 February 2023 |
| Creators | Marano, Jeffrey Matthew |
| Contributors | Graduate School, Paulson, Sally L., Weger-Lucarelli, James, Meng, Xiang-Jin, Aylward, Frank O. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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