Porcine reproductive and respiratory syndrome virus (PRRSV) is arguably the most economically-important swine pathogen. As the emergences of novel virulent strains of PRRSV continue to occur worldwide, rapid vaccine development is the key for effective control of ongoing PRRSV outbreaks. With the availability of the PRRSV reverse genetics systems, rapid vaccine development against PRRSV through the manipulation of the reverse genetics becomes feasible. To facilitate the vaccine development effort and study of PRRSV genes, we first established a DNA-launched infectious clone of the passage 14 PRRSV strain VR2385, pIR-VR2385-CA, and identified a spontaneous 435-bp deletion in the nsp2 gene. To characterize the biological and pathological significance of this nsp2 deletion, we restored deleted nsp2 sequence back to pIR-VR2385-CA and constructed another clone pIR-VR2385-R. VR2385-CA and VR2385-R were successfully rescued in vitro. The results from this study indicates that the spontaneous nsp2 deletion plays a role for enhanced PRRSV replication in vitro but has no significant effect on the pathogenicity of the virus. With the availability of the DNA-launched infectious clone of PRRSV, we successfully applied the molecular breeding approach to rapidly attenuate PRRSV. The GP5 envelope genes of 7 genetically divergent PRRSV strains and the GP5-M genes of 6 different PRRSV strains were molecularly bred. DS722 with shuffled GP5 genes and DS5M3 with shuffled GP5-M genes, were successfully rescued in vitro and shown to be attenuated both in vitro and in vivo. Furthermore, DS722, but not DS5M3, still elicit similar protection against PRRSV challenge as its parental virus. This study reveals a unique approach through DNA shuffling of viral envelope genes to attenuate a positive-strand RNA virus. We subsequently utilized the novel synthetic attenuated virus engineering (SAVE) approach to attenuate PRRSV. The GP5 and nsp9 genes of PRRSV were codon-pair deoptimized with the aid of a computer algorithm. SAVE5 and SAVE9 with deoptimized GP5 gene and SAVE9 with deoptimized nsp9 gene, were successfully rescued and shown to be attenuated in vitro. An in vivo pathogenicity study indicated the attenuation of SAVE5 virus in vivo. The results have important implications for rapid vaccine development against PRRSV and other important viruses. / Ph. D.

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Measles virus (MV) can cause severe acute diseases as well as long-lasting clinical deteriorations due to viral-induced immunosuppression and neuronal manifestation. How the virus enters the brain and manages to persist in neuronal tissue is not fully understood. Various mutations in the viral genes were found in MV strains isolated from patient brains. In this study, reverse genetics was used to introduce mutations in the fusion, matrix and polymerase genes of MV. The generated virus clones were characterized in cell culture and used to infect rat brain slice cultures. A mutation in the carboxy-terminal domain of the matrix protein (R293Q) promoted the production of progeny virions. This effect was observed in Vero cells irrespective of the expression of the signaling lymphocyte activation molecule (SLAM). Furthermore, a mutation in the fusion protein (I225M) induced syncytia formation on Vero cells in the absence of SLAM and promoted viral spread throughout the rat brain slices. In this study, a solid ex vivo model was established to elucidate the MV mutations contributing to neural manifestation.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23786号 / 医博第4832号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 髙折 晃史, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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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.

Arenaviruses cause an estimated 300,000 – 500,000 infections annually. Currently there is no arenavirus-specific antiviral drug available to treat these infections. This study sought to use the non-pathogenic New World arenavirus Tacaribe virus (TCRV) as a model for the pathogenic Junin virus (JUNV) and Machupo virus (MACV) that cause haemorrhagic fevers in South America. TCRV was used to explore three different approaches in the search for an antiviral drug against arenavirus infection targeted specifically against the viral nucleoprotein (NP). Of the four expressed arenaviral proteins, NP is the most abundant and is thought to be of multifunctional nature involved in viral replication, suppression of the innate immune system and viral egress. The approaches to find targets for broad-spectrum anti-arenaviral drugs were high throughput screens (HTS) with purified NP using thermal shift assays, exploring the virus interactions with the innate immune system and identifying virus- host protein-protein interactions. HTS resulted in the identification of two small- molecule compounds, [5-(2-Furyl)thien-2-yl]methanol and cyclosporine A (CsA), showing broad-spectrum activity against arenaviruses. Interferon-stimulated genes (ISGs), such as IFIT3, were identified to reduce viral titres and potential 202 protein- interactions between NP and host cell proteins were identified, of which the interaction with apoptosis-inducing factor 1 (AIF1) was described further. To characterise the importance of these interactions as potential drug targets further, a TCRV reverse genetics system was constructed.