The Pennsylvania State University The Graduate School Eberly College of Science COMPARATIVE GENOMICS PROVIDES INSIGHTS INTO HERPESVIRUS TRANSMISSION, SPREAD, AND VIRULENCE A Dissertation in Biochemistry, Microbiology, and Molecular Biology by Utsav Pandey Ó 2018 Utsav Pandey Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2018 The dissertation of Utsav Pandey was reviewed and approved* by the following: Moriah L. Szpara Assistant Professor of Biochemistry and Molecular Biology Dissertation Advisor Chair of Committee Andrew F. Read Evan Pugh Professor of Biology and Entomology Eberly Professor of Biotechnology Istvan Albert Research Professor Anthony Schmitt Associate Professor of Molecular Virology Director, Pathobiology Graduate Program Timothy C. Meredith Assistant Professor of Biochemistry and Molecular Biology Wendy Hanna-Rose Interim Department Head, Biochemistry and Molecular Biology Associate Professor of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School ii Abstract Herpesviruses are widespread in the nature infecting almost every animal species. They are significant pathogens for human health and agriculture. Herpes simplex virus 1 (HSV-1) causes millions of chronic infections in humans, whereas Marek’s disease virus 1 (MDV-1) is a herpesvirus of poultry of great economic importance. Clinical outcomes of HSV-1 infections are diverse, ranging from surface lesions, keratitis, to severe and potentially lethal encephalitis. Similarly, MDV-1 causes nervous system disease and lymphomas in chickens with mortality approaching 100% in absence of vaccination. Both HSV-1 and MDV-1 are alphaherpesviruses and contain a double-stranded DNA (dsDNA) genome as their genetic material. Using high throughput sequencing (HTS) and comparative genomics, this dissertation seeks to explore the genetic basis of transmission, spread and virulence of these viruses. In this dissertation, transmission of HSV-1 between individuals has been explored using transmission events between father-son and mother-neonate pair. Comparative genomics of these genomes showed that at the consensus level HSV-1 genomes can be identical even after multiple cycles of latency and reactivations. However, we observed that at the population level the parental and transmitted virus can be quite different. HSV-1 isolates obtained from the father- son pair were further characterized phenotypically using a murine animal model. By examining a series of phenotypic properties, we concluded that parental and transmitted viruses can also preserve their phenotypes over decades. Transmission and spread of MDV-1 in the field was depicted by sequencing viral isolates form poultry farms in central Pennsylvania. This study presented the first case of MDV-1 genomes obtained directly from chicken feathers and poultry dander without the use of cell culture. We showed that these genomes were highly identical at the consensus level, but differed at the population level, corroborating the findings from transmission events of HSV-1. This study was also important in laying foundation for studying MDV-1 genomics iii using field samples. We further extended comparative genomics of field samples of MDV-1 to understand the evolution of virulence in MDV-1. To this effect, we sequenced 64 spatially and temporally separated field isolates of MDV-1 and explored the role of recombination in emergence of highly virulent isolates over time. We found that paths to emergence of virulence in field isolates of MDV-1 is complex and could be explained by multiple recombination events between field isolates. We also employed comparative genomics to identify genetic markers of virulence in HSV-1 and MDV-1. In HSV-1, we identified amino acid variations in the viral protein VP22 that reliably distinguish low-virulence isolates from high- virulence isolates of HSV-1. To understand the biological importance of these amino acid residues in determining HSV-1 virulence, I have initiated the process of engineering viral mutants. These recombinant viral mutants can then be tested against the parental virus using in vitro and in vivo assays. Similarly, through comparison of field isolates of MVD-1 we have identified nucleotide substitutions that distinguish MDV-1 isolates of different pathotypes. Despite their widely recognized clinical and veterinary importance, there is a paucity of knowledge concerning the evolution and genetic basis of virulence for herpesviruses. Understanding the genetic diversity of parental and transmitted virus is invaluable to getting a comprehensive picture of a transmission event. Likewise, assessing the genetic diversity of clinical and field samples gives insights into the genetic variation in the circulating viral strains. Information obtained from these studies can be used to develop vaccines and therapeutics that account for the diversity present in the circulating strains. Similarly, identification of genetic markers of virulence can be used to gauge virulence level based on viral genotype providing a powerful tool for future diagnostics and prediction of clinical outcomes. Understanding how and why field isolates of MDV-1 became more pathogenic is of significance not just for poultry industry, but also in the wider context of combating increased incidence of drug and vaccine resistance among other pathogens. iv Table of contents LIST OF FIGURES ........................................................................................................ VIII LIST OF TABLES ......................................................................................................... XIV ACKNOWLEDGEMENT ................................................................................................ XV CHAPTER 1 ...................................................................................................................... 1 1.1 Genetic variation in pathogens and the advent of sequencing technologies ....... 2 1.2 Studying pathogen biology in the era of high throughput sequencing (HTS) ...... 3 1.3 Use of HTS in clinical diagnostics ....................................................................... 5 1.4 Use of HTS in epidemiological studies ................................................................ 7 1.5 Bioinformatics in clinical and public health laboratories ...................................... 9 1.6 Forward and reverse genetics approaches (Comparative genomics) ............... 11 1.7 Genetic variation in RNA vs. DNA viruses ......................................................... 12 1.8 Herpesvirus genetic variation and diversity ....................................................... 14 1.9 Alphaherpesviruses ........................................................................................... 16 1.10 Marek’s disease virus serotype 1 (MDV-1) ...................................................... 17 1.11 Herpes simplex virus-1 (HSV-1) ...................................................................... 18 CHAPTER 2 .................................................................................................................... 21 2.1 ABSTRACT ............................................................................................................... 22 2.2 IMPORTANCE ........................................................................................................... 23 2.3 INTRODUCTION ........................................................................................................ 24 2.4 MATERIALS AND METHODS ...................................................................................... 25 2.4.1 Collection of dust and feathers ....................................................................... 25 2.4.2 Viral DNA isolation from dust .......................................................................... 26 2.4.3 Isolation of viral DNA from feather follicles ..................................................... 28 2.4.4 Measurement of total DNA and quantification of viral DNA ............................ 29 2.4.5 Illumina next-generation sequencing .............................................................. 30 2.4.6 Consensus genome assembly ....................................................................... 31 2.4.7 Between-sample: consensus genome comparisons ...................................... 32 2.4.8 Within-sample: polymorphism detection within each consensus genome ...... 33 2.4.9 Testing for signs of selection acting on polymorphic viral populations ........... 33 2.4.10 Sanger sequencing of polymorphic locus in ICP4 ........................................ 34 2.4.11 Genetic distance and dendrogram ............................................................... 35 2.4.12 Taxonomic estimation of non-MDV sequences in dust and feathers ........... 35 2.4.13 GenBank accession numbers and availability of materials .......................... 35 2.5 RESULTS ................................................................................................................. 36 2.5.1 Sequencing, assembly and annotation of new MDV-1 consensus genomes from the field ............................................................................................................ 36 2.5.2 DNA and amino acid variations between five new field genomes of MDV-1 .. 39 2.5.3 Detection of polymorphic bases within each genome .................................... 40 2.5.4 Tracking shifts in polymorphic loci over time .................................................