Diseases in marine animals are emerging at an increasing rate. Disease forecasting enabled by virus surveillance presents a proactive solution for managing emerging diseases. Broad viral surveys aid in disease forecasting by providing baseline data on viral diversity associated with various hosts, including many that are not associated with disease. However, these viruses can become pathogens due to expansion in host or geographic range, as well as when changing conditions shift the balance between commensal viruses and the host immune system. Therefore, it is extremely valuable to identify and characterize viruses present in many different hosts in a variety of environments, regardless of whether the hosts are symptomatic or not. The lack of a universal gene shared by all viruses makes virus surveillance difficult, because no single assay exists that can detect the enormous diversity of viruses. Viral metagenomics circumvents this issue by purifying viral particles directly from host tissues and sequencing the nucleic acids, allowing for virus identification. However, virus identification is only the first step, which should ideally be followed by complete sequencing of the viral genome to identify genes of interest and develop assays to reveal viral prevalence, tropism, ecology, and pathogenicity. This dissertation focuses on the discovery of novel viruses in marine animals, characterization of complete viral genomes, and the development of subsequent diagnostic assays for further analysis of virus ecology. First, viral metagenomics was used to explore the viruses present in the healthy Weddell seal (Leptonychotes weddellii) population in Antarctica, which led to the discovery of highly prevalent small, circular single-stranded DNA (ssDNA) viruses. The lack of knowledge regarding the viruses of Antarctic wildlife warrants this study to determine baseline viral communities in healthy animals that can be used to survey changes over time. From the healthy Weddell seals, viral metagenomics led to the discovery of 152 novel anellovirus genomes, encompassing two anellovirus species. Characterizing these viruses is important for understanding the prevalence and diversity of ssDNA viruses, which have only recently been described in marine animals. Furthermore, since emerging diseases can be caused by changing conditions affecting host susceptibility to a virus that was previously not related to disease (opportunistic pathogen), having baseline data allows for quick identification of the pathogen. In addition to determining baseline data, viral metagenomics can explore the role of viruses in disease. A novel virus, Asterias forbesi-associated circular virus (AfaCV), was discovered in the Atlantic sea star Asterias forbesi displaying symptoms of sea star wasting disease (SSWD). AfaCV was the first circular replicase-encoding ssDNA (CRESS-DNA) virus discovered in echinoderms, but it was only present in 10% of SSWD sea stars indicating it is not involved in the development of the disease. This dissertation also focuses on elucidating the role of two previously characterized viruses, chelonid fibropapillomatosis-associated herpesvirus (CHHV5; Chelonid herpesvirus 5, ChHV5) and Zalophus californianus anellovirus (ZcAV), in animal health. PCR amplicon sequencing was used to obtain large portions of the 132 kb genome of ChHV5, the putative etiological agent of the neoplastic sea turtle disease, fibropapillomatosis. Obtaining the genome of ChHV5 from Florida green, Kemp’s ridley, and loggerhead sea turtles provides data for phylogenetic analysis across geographic locations and sea turtle species, as well as a reference for designing downstream molecular assays to examine viral latency. ZcAV was first described from the lungs of captive sea lions involved in a mortality event. PCR could not detect ZcAV in the blood of infected animals, and since sea lions are a protected species, it is not possible to obtain lung biopsies from live sea lions to determine ZcAV prevalence or its role in sea lion health. To answer these important questions, an enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to ZcAV in serum from wild sea lion populations. This newly developed ELISA showed that sea lions mount an immune response to ZcAV, and was used to determine the prevalence of ZcAV among wild sea lion populations. This dissertation makes an important contribution to marine science through discovery and characterization of viruses present in healthy and diseased marine animals. Several different methods were used for virus whole-genome sequencing including viral metagenomics, PCR amplicon sequencing, and target enrichment. These findings were expanded upon by developing and using PCR assays and a serological assay to screen for virus prevalence. These methods have implications for viral surveillance and understanding the role of novel viruses in animal health.

The pathogen Campylobacter fetus (CF) is classified into two subspecies, Campylobacter fetus subspecies fetus (CFF) and Campylobacter fetus subspecies venerealis (CFV). Even though CFF and CFV are genetically closely related, they exhibit differences in their host adaptation; CFF inhabits the gastrointestinal tract of both humans and several animal species, while classical CFV is specific to the bovine genital tract and is of particular concern with respect to international bovine trade regulation. Traditionally, differentiation between the two subspecies has been achieved using a limited number of biochemical tests but more rapid and definitive genetic methods of discrimination are desired. A recent study suggested that the presence of a genomic island only in CFV could discriminate between the two sub- species but this hypothesis could not be confirmed on a collection of isolates originating in Canada. To identify alternative gene targets that would support accurate subspecies discrimination, this study has applied several approaches including suppression subtractive hybridization and whole genome sequencing supplemented with optical mapping. A subtractive hybridization screen, using a well-characterized CFV isolate recovered during routine screening of bulls in an Artificial Insemination center in western Canada and that lacked much of the genomic island and a typical Canadian CFF isolate, yielded 50 clones; characterization of these clones by hybridization screening against selected CF isolates and by nucleotide sequence BLAST analysis identified three potentially CFV-specific clones that contained inserts originating from a second genomic island. Further screening using a larger CF sample set found that only Clone #35 was truly CFV-specific. Optical maps (NcoI digest) of the Canadian CFF and CFV isolates used for the subtractive hybridization showed that certain regions of these genomes were quite distinct from those of two reference strains. Whole genome sequencing of these two isolates identified two target genes (PICFV5_ORF548 and CFF_Feature #3) that appear to be selectively retained in the two subspecies. Screening of a collection of CF isolates by PCRs targeting these three loci (SSH_Clone #35, PICFV5_ORF548 and CFF_Feature #3) supported their use for subspecies discrimination. This work demonstrates the complex genomic diversity associated with these CF subtypes and the challenge posed by their discrimination using limited genetic loci.

Thauera aminoaromatica MZ1T, a floc-forming bacterium isolated from an industrial activated sludge wastewater treatment plant, overproduces exopolysaccharide (EPS) leading to viscous bulking. This phenomenon results in poor sludge settling and dewatering during the clarification process. To identify genes responsible for bacterial flocculation, a whole genome phenotypic sequencing technique was applied. Genomic DNA of MZ1T flocculation-deficient mutants were subjected to massively parallel sequencing. The resultant high-quality reads were assembled and compared to the reference genome of the wild type genome. We identified nine nonsynonymous mutations and one nonsense mutation putatively involved in EPS biosynthesis. Complementation of the nonsense mutation located in an EPS deacetylase gene restored the flocculating phenotype. The FTIR spectra of EPS isolated from the wild-type showed reduced C=O peak of the N-acetyl group at 1665 cm-1 as compared to the spectra of MZ1T floc-deficient mutant EPS, suggesting that the WT EPS was partially deacetylated. Gene expression analysis also demonstrated the deacetylase gene transcript increased before flocculation occurred. The results suggest that the deacetylation of MZ1T EPS is crucial for flocculation. The information obtained from this study will be useful for preventing viscous bulking and wastewater treatment system failure, and may have potential applications in the biotechnology sector for the controlled removal of cells.

In healthcare settings and beyond, the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) among other pathogens exchange antibiotic resistance and virulence factors and emerge as new infectious clones. According to the Saudi General Authority for Statistics (stats.gov.sa), Saudi Arabia is a country where more than 27 million pilgrims meet in annual continual mass-gathering events. This massive influx of people could introduce novel pathogens to the community that could not necessarily be detected with traditional culture-dependent clinical microbiological tests. Conventional clinical microbiology and environmental pathogen detection methods have had many limitations and narrow search scope. These methods can only target known and culturable pathogens. Over the past decade, applications of next-generation sequencing (NGS) and bioinformatics tools have revolutionized the way pathogens are detected and their relevant phenotypes such as clonal types, antibiotic resistance are predicted to aid in clinical decision making as additional practice to traditional clinical microbiology-based testing protocols. The aim of this study was to apply whole-genome sequencing (WGS) and bioinformatic analysis tools on clinical samples and bacterial isolates in order to pave the way for transforming current clinical microbiology practices in a tertiary referral hospital in Jeddah, Saudi Arabia. My attempt to utilize WGS as a tool on pathogenic strains in this study combined with the clinical data has resulted in discovering a silent outbreak of an emerging hypervirulent strain of Klebsiella pneumoniae (Chapter 2). Analysis of the strains antimicrobial profiles genetically has yielded the first characterization of a misidentified Klebsiella quasipneumoniae harboring plasmid-mediated carbapenemases of Klebsiella pneumoniae carbapenemases (KPC) (Chapter 3). Similarly, I was able to study mobile colistin resistance genes in the isolates and identify a novel occurrence of mcr-1 and mcr-8 (Chapter 4). I applied clinical metagenomic protocol on an intestinal biopsy of an inflammatory bowel disease patient with Crohn’s disease, where I identified an association of three co-occurring and an actively replicating non-tuberculosis mycobacteria (Chapter 5). The deployment of whole-genome sequencing and metagenomic in infectious disease surveillance and diagnostics could prove beneficial in limiting epidemics and detect transmission patterns of antimicrobial-resistant genes.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22762号 / 工博第4761号 / 新制||工||1745(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 田中 宏明, 教授 米田 稔, 准教授 松田 知成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Amaranthus hypochondriacus is an emerging pseudo-cereal native to the New World which has garnered increased attention in recent years due to its nutritional quality, in particular its seed protein, and more specifically its high levels of the essential amino acid lysine. It belongs to the Amaranthaceae family, is an ancient paleotetraploid that shows amphidiploid inheritance (2n=32), and has an estimated genome size of 466 Mb. Here we present a high-quality draft genome sequence of the grain amaranth A. hypochondriacus. The genome assembly consisted of 377 Mb in 3,518 scaffolds with an N50 of 371 kb. Repetitive element analysis predicted that 48% of the genome is comprised of repeat sequences, of which Copia-like elements were the most common classified retrotransposon. A transcriptome, consisting of 66,370 contigs, was assembled from eight different tissue and abiotic stress libraries. Annotation of the genome identified 23,059 genes that were supported by our de novo transcriptome assembly, the RefBeet 1.1 gene index and the Uniprot_sprot database. To describe the genetic diversity within the grain amaranths (A. hypochondriacus, A. caudatus, and A. cruentus) and their putative progenitor (A. hybridus) we re-sequenced seven accessions in the genus Amaranthus (four A. hypochondriacus, and one of each A. caudatus, A. cruentus, and A. hybridus), which identified 7,184,636 and 1,760,433 interspecific and intraspecific single nucleotide polymorphisms, respectively. A phylogeny analysis of the re-sequenced accessions substantiated the classification of A. hybridus as the progenitor species of the grain amaranths. Lastly, we generated a physical map for A. hypochondriacus using the BioNano optical mapping platform. The physical map spanned 340 Mb and a hybrid assembly using the BioNano optical genome maps nearly doubled the N50 of the assembly to 697 kb. Moreover, we analyzed synteny between amaranth and Beta vulgaris (sugar beet) and estimated, using Ks analysis, the age of the most recent polyploidization event in amaranth.

Chenopodium quinoa, a South American pseudocereal, has valuable agricultural traits such as salt tolerance and drought tolerance, and it has beneficial nutritional properties such as high protein content and a complete amino acid profile. However, problems including disease susceptibility, low harvest index, lodging, seed shattering, low heat tolerance, and saponin content plague quinoa. Genetic resources for quinoa are needed to fix these problems and make quinoa more available throughout the world. We used ethyl methanesulfonate (EMS) to create a mutant population of QQ74 quinoa (USDA GRIN PI 614886) of 5,030 mutant families. We did whole exome sequencing (WES) on 44 mutant families. Using the recently published quinoa reference genome and MAPS, a mutation detection pipeline, we found a mutation rate of 11.35 mutations/Mb in these families. We also used whole genome sequencing (WGS) to calculate a mutation rate of 21.67 mutations/Mb in an additional nine mutant families. To demonstrate the utility of this population as a genetic resource, we found an EMS-induced nonsense mutation in the betalain synthesis pathway that prevents red betacyanins from accumulating in the hypocotyl of quinoa. With the mutation rates in our population, we calculate that analysis of 300 mutant families will yield 3-7 mutations in any gene of interest, which will facilitate forward and reverse genetic studies in quinoa.

Inherited Retinal Degenerations (IRDs) are a heterogenous group of diseases which lead to vision impairment and can be found both in humans and in dogs. About 1 in 1,380 humans is estimated to suffer from an autosomal recessive IRD, which would be 5.5 million people worldwide, and many more are estimated to be unaffected carriers. This makes autosomal recessive IRDs likely the most common group of Mendelian diseases in humans. Today, about 300 genetic mutations have been connected to cause retinal diseases in humans. Whilst in dogs only 32 genes have been identified, numerous eye conditions have been described where the genetic cause has not yet been identified. This suggests that there are much more genetic causes to discover in the dog genome. Additionally, the dog serves well as a model organism to investigate IRDs as it is sharing morphological and genetic similarities with humans. For these reasons, proper software, a canine reference genome of high quality, and smart implementation of bioinformatic tools and methods are a big advantage to increase chances of finding new causative genetic variants and subsequently enable faster detection of possible preventions of the disease or at least alleviating its symptoms via early diagnosis. In this project, a pre-existing pipeline consisting of Bash scripts was stepwise improved with the goal to increase its efficiency. After controlling whether previous data could still be reproduced with the old pipeline in a first step, the software was exchanged to more updated versions in a second step. A main change was the replacement of the mapping tool Burrows-Wheeler Aligner (BWA) from bwa mem to bwa-mem2 mem, and the update of deprecated Genome Analysis Toolkit (GATK) 3.7 to version 4.3 or 4.4. Thirdly, the scripts were adapted from using the older canine reference genome CanFam3.1 to CanFam4. In a fourth step, for automatization and fastening the running time, the pipeline steps were implemented into the workflow management system Nextflow. Additionally, this step was partly aiming to make the pipeline in concordance with the FAIR-principles. All steps were tested on the same test data set, a Labrador retriever family trio, in which one genetic cause for a canine form of the IRD Stargardt disease in a previous study had been detected, namely an insertion in the ABCA4 gene. Lastly, the workflow was also tested on a second data set of a novel IRD of unknown genetic origin on two sibling pairs of Chinese Crested Dogs (CCR). The adjustment of the pipeline shows similar results regarding the change of mapping tool. Introducing the new reference genome revealed a drop of average coverage by one read average for when using CanFam4, while other results were similar. Using the new reference genome increased the number of unknown variants compared to findings with CanFam3.1. However, the known causative variant for the canine form of Stargardt disease, an insertion in ABCA4 gene, could be found in all cases. The run with Nextflow produced identical results to when the respective steps were run with Bash scripts, but it reduced the running time. Running the workflow on the new data set (CCR) and subsequent annotation and filtering indicate new candidates which could be further investigated as a potential cause for this currently unknown cause for an IRD.

The Scandinavian brown bear went through a severe bottleneck due to human actions in the early 1900’s that reduced the population to low numbers. After protective measures were taken, the population recovered and today it consists of around 3000 individuals. Such loss of genetic variation can have long-lasting effects on population viability even as populations recover, and is therefore important to consider in the management and conservation of species. Previous studies investigating the genetic effects of the bottleneck based on genetic markers, such as microsatellites and mitochondrial DNA, have rendered inconclusive results. Here, whole-genome sequencing of historical and contemporary bears was used to estimate heterozygosity, inbreeding and mutational load in the pre- and post- bottleneck population. Surprisingly, a significant increase in heterozygosity was found in the contemporary population and no significant increase in inbreeding over time was found. However, mutational load was higher in the contemporary subpopulation in the south of Sweden compared to the subpopulation in the north, and the southern subpopulation seems to have lost unique genetic variation after the bottleneck. The results indicate that although the population was negatively affected by the bottleneck, a following increase in gene flow could have contributed to successful recovery of the population.

Strains MS455 and MS389, endophytic bacteria, were isolated from healthy soybean plant growing adjacent to a patch of plants affected by charcoal rot disease, caused by the fungal pathogen Macrophomina phaseolina. The complete genomes of both strains were sequenced and identified as Burkholderia species Strain MS455 exhibits broad-spectrum antifungal activities against economically important pathogens, including Aspergillus flavus. Random and site-specific mutations were employed in discovery of the genes that share high homology to the ocf gene cluster of Burkholderia contaminans strain MS14, which is responsible for production of the antifungal compound occidiofungin. RNA-seq analysis demonstrated ORF1, a homolog to the ambR1 LuxR-type regulatory gene, not only regulates occidiofungin biosynthesis in MS455, but also involved in expression of multiple genes, especially those involved in ornibactin biosynthesis. Plate and corn kernel assays showed that growth of A. flavus and aflatoxin production were reduced significantly by MS455 as compared with buffer control and the ORF1 mutant. Strain MS389 shows significant antifungal and antibacterial activities as well. Mutagenesis study identified that the TatC gene, an important unit of twin-arginine translocase (Tat) secretion system, and the LysR-type transcriptional regulatory gene were essential for the antifungal activity of strain MS389. RNA-seq analysis implied that the pyrrolnitrin biosynthesis gene cluster and an uncharacterized NRPS / PKS gene cluster were involved in antifungal activity. By comparing several endophytic bacteria of Burkholderia, including MS455 and MS389, to pathogenic Burkholderia species, endophytic bacteria were observed to harbor multiple antimicrobial biosynthesis genes but lack certain pathogenic or virulence genes. The potential endophytic behavior related genes and characteristics related to antibiotic resistance, secretion system, and CRISPR-Cas profiles were well established. The research findings on strains MS455 and MS389 have provided important genetic clues for understanding their molecular mechanism of antimicrobial activities and exhibited their potential possibility as biocontrol agents.