A phylogenomic analysis of Marek's disease virus reveals independent paths to virulence in Eurasia and North America

Virulence determines the impact a pathogen has on the fitness of its host, yet current understanding of the evolutionary origins and causes of virulence of many pathogens is surprisingly incomplete. Here, we explore the evolution of Marek's disease virus (MDV), a herpesvirus commonly afflicting chickens and rarely other avian species. The history of MDV in the 20th century represents an important case study in the evolution of virulence. The severity of MDV infection in chickens has been rising steadily since the adoption of intensive farming techniques and vaccination programs in the 1950s and 1970s, respectively. It has remained uncertain, however, which of these factors is causally more responsible for the observed increase in virulence of circulating viruses. We conducted a phylogenomic study to understand the evolution of MDV in the context of dramatic changes to poultry farming and disease control. Our analysis reveals evidence of geographical structuring of MDV strains, with reconstructions supporting the emergence of virulent viruses independently in North America and Eurasia. Of note, the emergence of virulent viruses appears to coincide approximately with the introduction of comprehensive vaccination on both continents. The time-dated phylogeny also indicated that MDV has a mean evolutionary rate of ~1.6 × 10−5 substitutions per site per year. An examination of gene-linked mutations did not identify a strong association between mutational variation and virulence phenotypes, indicating that MDV may evolve readily and rapidly under strong selective pressures and that multiple genotypic pathways may underlie virulence adaptation in MDV

Molekularer Mechanismus der Codonpaardeoptimierung zur Attenuierung von Viren

The chemical synthesis of nucleic acids enables the generation of recoded genes with novel properties. The synonymous genome recoding introduces a large number of mutations into recoded genes without changing the amino acid composition of encoded proteins. The goal of the recoding is to alter codon usage, codon pair usage or dinucleotide frequencies of the recoded genes and thereby change different biological properties. Codon optimization and codon pair optimization are used to improve protein production from recoded genes. On the contrary, the goal of codon deoptimization and codon pair deoptimization is to reduce the protein expression of recoded genes, which can be used for the production of experimental live-attenuated virus vaccine candidates. While codon and codon pair deoptimization are capable of producing attenuated viruses, the molecular basis behind the attenuation is not well understood. In my work, I confirmed that codon and codon pair deoptimization are both excellent tools for virus attenuation, and therefore suitable for the development of viral vaccines. These live-attenuated virus vaccines, prepared by both methods, induced in mice model a remarkable protection against WT virus challenge. The main goal of this project was to elucidate the molecular mechanism of virus attenuation by codon pair deoptimization. Due to low frequencies of CpG dinucleotides in the human genome, codon pair deoptimization unintentionally increases the number of CpG dinucleotides in the recoded genes. Therefore, it was assumed that CpG dinucleotides have a substantial impact on virus attenuation. I showed that increased numbers of CpG dinucleotides do not influence influenza A virus. However, I discovered that RNAs produced by codon pair deoptimized genes have higher degradation rates. Additionally, I found out that codon pair deoptimization reduces protein production of recoded genes also by decreasing their translation efficiency. These findings elucidate the highly complex molecular mechanism underlying the synonymous genome recoding. In the second part of my work, I showed that the commonly used codon optimization is a suitable method to increase the protein expression of target genes. More importantly, I demonstrated that also codon pair optimization has a great potential to improve RNA stability and protein production. Therefore, codon pair optimization might be a new powerful tool to enhance protein expression for diverse applications in biotechnology

DEEPGEN TM-A novel variant calling assay for low frequency variants

Detection of genetic variants in clinically relevant genomic hot-spot regions has become a promising application of next-generation sequencing technology in precision oncology. Effective personalized diagnostics requires the detection of variants with often very low frequencies. This can be achieved by targeted, short-read sequencing that provides high sequencing depths. However, rare genetic variants can contain crucial information for early cancer detection and subsequent treatment success, an inevitable level of background noise usually limits the accuracy of low frequency variant calling assays. To address this challenge, we developed DEEPGENTM, a variant calling assay intended for the detection of low frequency variants within liquid biopsy samples. We processed reference samples with validated mutations of known frequencies (0%-0.5%) to determine DEEPGENTM's performance and minimal input requirements. Our findings confirm DEEPGENTM's effectiveness in discriminating between signal and noise down to 0.09% variant allele frequency and an LOD(90) at 0.18%. A superior sensitivity was also confirmed by orthogonal comparison to a commercially available liquid biopsy-based assay for cancer detection

Novel Divergent Polar Bear-Associated Mastadenovirus Recovered from a Deceased Juvenile Polar Bear

Cross-species transmission of viral pathogens is becoming an increasing problem for captive-animal facilities. This study highlights how animals in captivity are vulnerable to novel opportunistic pathogens, many of which do not result in straightforward diagnosis from symptoms and histopathology. In this study, a novel pathogen was suspected to have contributed to the death of a juvenile polar bear. HTS techniques were employed, and a novel Mastadenovirus was isolated. The virus was present in both the tissue and blood samples. Phylogenetic analysis of the virus at both the gene and genome levels revealed that it is highly divergent to other known mastadenoviruses. Overall, this study shows that animals in isolated conditions still come into contact with novel pathogens, and for many of these pathogens, the host reservoir and mode of transmission are yet to be determined.Polar bears in captivity can be exposed to opportunistic pathogens not present in their natural environments. A 4-month-old polar bear (Ursus maritimus) living in an isolated enclosure with his mother in the Tierpark Berlin, Berlin, Germany, was suffering from severe abdominal pain, mild diarrhea, and loss of appetite and died in early 2017. Histopathology revealed severe hepatic degeneration and necrosis without evidence of inflammation or inclusion bodies, although a viral infection had been suspected on the basis of the clinical signs. We searched for nucleic acids of pathogens by shotgun high-throughput sequencing (HTS) from genomic DNA and cDNA extracted from tissue and blood. We identified a novel Mastadenovirus and assembled a nearly complete genome from the shotgun sequences. Quantitative PCR (qPCR) revealed that viral DNA was present in various concentrations in all tissues examined and that the highest concentrations were found in blood. Viral culture did not yield cytopathic effects, but qPCR suggested that virus replication was sustained for up to three passages. Positive immunofluorescence staining confirmed that the virus was able to replicate in the cells during early passage. Phylogenetic analysis demonstrated that the virus is highly divergent compared to other previously identified Mastadenovirus members and basal to most known viral clades. The virus was found only in the 4-month-old bear and not in other captive polar bears tested. We surmised, therefore, that the polar bear was infected from an unknown reservoir, illustrating that adenoviral diversity remains underestimated and that cross-species transmission of viruses can occur even under conditions of relative isolation

Data from: A phylogenomic analysis of Marek's disease virus (MDV) reveals independent paths to virulence in Eurasia and North America

Virulence determines the impact a pathogen has on the fitness of its host, yet current understanding of the evolutionary origins and causes of virulence of many pathogens is surprisingly incomplete. Here, we explore the evolution of Marek's disease virus (MDV), a herpesvirus commonly afflicting chickens and rarely other avian species. The history of MDV in the 20th century represents an important case study in the evolution of virulence. The severity of MDV infection in chickens has been rising steadily since the adoption of intensive farming techniques and vaccination programs in the 1950s and 1970s respectively. It has remained uncertain, however, which of these factors is causally more responsible for the observed increase in virulence of circulating viruses. We conducted a phylogenomic study to understand the evolution of MDV in the context of dramatic changes to poultry farming and disease control. Our analysis reveals evidence of geographical structuring of MDV strains, with reconstructions supporting the emergence of virulent viruses independently in North America and Eurasia. Of note, the emergence of virulent viruses appears to coincide approximately with the introduction of comprehensive vaccination on both continents. The time-dated phylogeny also indicated that MDV has a mean evolutionary rate of ~1.6 x 10-5 substitutions / site / year. An examination of gene-linked mutations did not identify a strong association between mutational variation and virulence phenotypes, indicating that MDV may evolve readily and rapidly under strong selective pressures, and that multiple genotypic pathways may underlie virulence adaptation in MDV

R code file

Contains R code and paths to relevant files in the data packag

phylogenetic analysis

Contains whole genome alignments, BEAST and PhyML file

mutation analysis

Contains FastML output files and ORF mutation informatio

The Anti-amyloid Compound DO1 Decreases Plaque Pathology and Neuroinflammation-Related Expression Changes in 5xFAD Transgenic Mice

Self-propagating amyloid-β (Aβ) aggregates or seeds possibly drive pathogenesis of Alzheimer's disease (AD). Small molecules targeting such structures might act therapeutically in vivo. Here, a fluorescence polarization assay was established that enables the detection of compound effects on both seeded and spontaneous Aβ42 aggregation. In a focused screen of anti-amyloid compounds, we identified Disperse Orange 1 (DO1) ([4-((4-nitrophenyl)diazenyl)-N-phenylaniline]), a small molecule that potently delays both seeded and non-seeded Aβ42 polymerization at substoichiometric concentrations. Mechanistic studies revealed that DO1 disrupts preformed fibrillar assemblies of synthetic Aβ42 peptides and decreases the seeding activity of Aβ aggregates from brain extracts of AD transgenic mice. DO1 also reduced the size and abundance of diffuse Aβ plaques and decreased neuroinflammation-related gene expression changes in brains of 5xFAD transgenic mice. Finally, improved nesting behavior was observed upon treatment with the compound. Together, our evidence supports targeting of self-propagating Aβ structures with small molecules as a valid therapeutic strategy