The study of rotavirus phylogenetic diversity, re- assortment and interspecies transmission and associated virome in Northern Irish livestock exotic animals and wildlife Paula Lagan Tregaskis1,2, Mark Mooney2 and Ken Lemon1 1Virology Branch, Agri-food and Biosciences Institute Northern Ireland, Stoney Road, Stormont, Belfast BT4 3SD, UK 2Institute for Global Food Security, Biological Sciences, Queens University Belfast 3) VISUALISATION INTRODUCTION 1) DATA COLLECTION & QUALITY PROCESSING RESULTS 3b Korna chart Rotavirus (RV) is a highly infectious pathogen responsible for • Expected overall depth of reads expected at 25 2) WORKFLOW DATA ANALYSIS Porcine Microbiome WTA 79 acute diarrhoea in infants and animals worldwide. Enteritis is million the most common cause of mortality in neonatal calves in • Libraries yields per pair-end read; 500,000 to Northern Ireland. 3.4 million per sample. ANALYSIS A: ANALYSIS B: EDGE Platform AFBI Veterinary Science Division identified rotavirus as the Empowering the Development of Genomics Expertise • 97% Q30 (1 in 1000 base called incorrectly Trimmomatic MRC CLIMB project (Cloud Infrastructure for Microbial Bioinformatics) causative agent of enteritis in 28% of young calves and 48% 99.9%) of piglets faecal samples. • Remove low complexity reads, short reads and Meta-SPADES-Assembly uses k-mers for • RV is classified into groups from Group A to I and has a reads containing N building the initial de Bruijn graph EDGE pre-processing trimming 18Kb double stranded RNA genome. Group A is the most • Remove adaptors trim poor quality reads prevalent and virulent rotavirus. • Match pair-ends reads BLAST n and BLAST X alignment SPADES-Assembly uses k-mers for • Rotavirus has a dsRNA segmented genome with 11 for taxonomic classification building the initial de Bruijn graph segments. • Genotyping is based on the dual viral outer proteins VP7 MEGAN (MEtaGenome ANalyser) to explore Taxonomy classification using and visualise taxonomic groups multiple tools including BWA mapping, Kraken glycoprotein (G) and VP4 protease sensitive and GOTTCHA hemagglutinin [P] or whole genome classification • New RV strains are classified after submission to the 3a MEGAN -Bovine WTA 18 Rotavirus classification working group. AIM of STUDY 1) Complete a molecular detection rotavirus study in Northern Ireland livestock, exotic animals and wildlife. 2) Phylogenetic diversity study of the genotype rotavirus group A. 3) Investigation of the associated enteric virome of clinically diseased animals to detect possible co-pathogens. EDGE Pre-processing 4) Identify if re-assortment and interspecies transmission occurs. Porcine Virome WTA 79 MATERIALS and METHODS RESULTS – Snapshot of enteric virome and RV phylogenetics 1) Sample biobank and screening for Porcine microbiome Bovine & Ovine Virome Equine, wildlife & exotic microbiome RV VP6 gene n=326 clinical disease faecal sample Samples Screening VP6 gene RVA by qPCR assay Selected 52 symptomatic negative samples Selected 39 VP6 RVA positive samples 2) Next Generation Sequencing (NGS) 2a) Viral Enrichment method: Rotavirus genotyping results RNA viral genomes by whole transcriptome amplification (WTA) DNA viral genome by whole genome amplification (WGA) Pudu RV phylogenetics 2b) Library preparation method: *Rotavirus Classification Working group submission for further verification and genotype assignment CONCLUSIONS and FUTURE WORK The faecal virome of enterically diseased animals is dominated by dsDNA bacteriophage Caudovirales order and families Siphovirdae, Myoviridae and Podoviridae as demonstrated in the porcine WTA 79 Korna virome chart and heat maps. Rotavirus is the most abundant virus of these clinically diseased livestock animals and some wildlife virome alongside the bacteriophages and coexists with other enteric viruses. The preliminary mapping of reads shows other viruses present in the enteric virome which is dependant on animal species. Most commonly found viruses observed with rotavirus were ungulate bocaparvovirus, aichivirus, astrovirus, mastadenovirus A and stool-associated circular virus. Insect and plant viruses were also shown in viromes of animals indicative of ingested viruses from their diet. 2c) NGS run method: Preliminary phylogenetics of RVA found common genotypes were present in porcine samples such as G3 P[13] and bovine G6 P[11] however novel strains of RVA • 5 MiSeq (Illumina) runs -V3 600 Cycles Viral metagenome were also present in bovine, pudu and ovine samples. It remains unclear if rotavirus inter-species transmission or reassortment has occurred at this stage of denovo pair-end reads. analysis although the pudu strain of RVA is most similar to a human strain. • Bioinformatics programs for quality trim and to filter Future work will be the continual review of analysis NGS bioinformatics pipelines for suitable semi-quantitative, sensitive and accurate analysis that represents the reads, assembly, align and taxonomically classify before virome samples. Reference genome mapping of rotavirus and a comprehensive phylogenetic mapping from all animal species in this study will be completed to visualisation. demonstrate the diversity of rotavirus within the Northern Ireland region. Lastly, unclassified reads will be studied to identify possible new viruses. .