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Holmes OHA 2017 Redefining the Virosphere: Metagenomics & Disease Emergence Eddie Holmes Marie Bashir Institute for Infectious Diseases and Biosecurity, Charles Perkins Centre, School of Life & Environmental Sciences and Sydney Medical School, The University of Sydney, Australia Understanding Disease Emergence Infectious disease emergence is often due to host-jumping of pathogens Questions • How do viruses adapt to and spread in new hosts? • How large is the virosphere and how is it structured? • What determines virus diversity and abundance? ‘Fault lines’ in Emergence We characterize virus biodiversity and evolution, especially in locations where humans and animals interact PUUV 1.00/100 SA14 SANGV SA22 1.00/100 1.00/100 Slo/Af-BER DOBV Tracking Viral Emergence DOBV/Ano-Poroia/Af19/1999 1.00/100 Saaremaa-160V SAAV 0.73/100 Aa1854 SK/Aa 1.00/100 Jurong TJK/06(RT49) SERV THAIV-741 THAIV 80-39 0.99/100 1.00/100 L99 XXA001 SEOV BjHD01 1.00/100 Z37 1.00/100 YongjiaRf45 YongjiaRn14 Gou3 1.00/100 ZJ5 1.00/100 LongquanRn-08-72 LongquanHu-201005 1.00/100 LongquanRn-08-101 LongquanRn-09-642 GOUV 0.97/100 LongquanRn-08-712 LongquanRn-09-132 LongquanRn-09-92 LongquanRf-11-163 LongquanRn-11-230 LongquanRn-11-212 LongquanRf-11-122 LongquanRn-11-1 LongquanRn-11-220 1.00/100 NC167 DBSV Wencheng-Nc-427 1.00/100 JilinAP06 ASV 1.00/100 SC1 76-118 1.00/100 Bao14 1.00/100 CGAa1011 ZLS-12 0.87/100 A9 1.00/100 CGRn5310 84FLi 0.74/100 Q32 Z5 0.99/100 Hantaviruses LongquanHu-200938 HTNV 1.00/100 LongquanHu-201123 Frequent spillover from LongquanAa-08-150 1.00/100 LongquanHu-201010 rodents to humans but no LongquanHu-201039 LongquanAa-09-24 LongquanAa-09-698 onward human-to-human LongquanAa-08-196 LongquanAa-08-492 transmission (evolutionary LongquanMf-09-8 LongquanAa-09-238 LongquanMf-09-473 dead-end) LongquanAa-10-101 LongquanAa-10-393 0.01 LongquanAa-10-74 Meta-Transcriptomics (Bulk RNA-Seq) • Sample processing • Homogenize samples from representative tissue • Extract total RNA • Pool samples • Library preparation • Deplete ribosomal RNA (rRNA) • Prepare cDNA library • High-throughput sequencing on Illumina platform • Target ~50 millions reads per library • Sequencing all ‘non-ribosomal’ RNA in sample • Confirmation by PCR and Sanger sequencing Transcriptome Data Analysis Pipeline • Remove host data by mapping against reference (optional) • De novo assemble transcriptome – Trinity or any decent assembler – Estimate abundance/frequency of transcripts • Compare contigs against sequence databases using BLAST – Blastn: simple and fast for known pathogens – Blastx: slower but better for novel pathogens – CD search: find gene homologs • Refine assembly and annotate hits Mang Shi, J-S Eden – Phylogenetic analysis (using the RdRp as a marker) • These data provide an unbiased view of the virome/pathogen discovery Sample quality is critical, including rapid storage at -80ºc. redefining the virosphere Picorna Hepe- Nido Reo -Calici Virga Extraordinary RNA Nidovirales Virus Diversity in Narna- Levi Reoviridae Invertebrates Bunya-Arena Virgaviridae, Bromoviridae, Closteroviridae, Togaviridae, • Collaboration with the Chinese Endornaviridae, Tymovirales, Hepeviridae, Alphatetraviridae CDC, Beijing Tombus- Ourmiavirus, • Examined >220 species across 9 Noda Narnaviridae, Leviviridae invertebrate phyla Partiti- Picobirna • Huge data (>100 libraries, >3 Tbp) • Overall ~1500 newly discovered Bunyaviridae, Tenuiviurs, Arenaviridae, Emaravirus Mono-Chu viruses in diverse invertebrate hosts (new genera, families, orders) • Invertebrate viruses often ancestral Partitiviridae, Picobirnaviridae to vertebrate viruses Tombusviridae, Umbravirus, Toti-Chryso Luteoviridae (Luteovirus), • Fills in ‘gaps’ in phylogenetic Nodaviridae Luteo- diversity – taxonomic implications Sobemo • Combination of virus-host co- divergence and cross-species virus Mononegavirales, Chuvirus, Ophiovirus, New -ve viruses transmission Totiviridae, Chrysoviridae, Quadriviridae Orthomyxo Permutotetra • Red = newly described here Grey = known viruses Picornavirales Luteoviridae (Polerovirus & Caliciviridae Emanovirus), Sobemovirus Permutotetraviridae Orthomyxoviridae “Orthomyxo-Like” Virus Phylogenetic Diversity 100 AJG39090 Wuhan Louse Fly Virus 4 [CC63432] (ref) 0.5 99 #AJG39089 Wuhan Louse Fly Virus 3 [CC56639] (ref) 87 AJG39084 Jingshan Fly Virus 1 [fly116939] (ref) AJG39088 Shuangao Insect Virus 4 [insectZJ68168] (ref) 97 #AJG39086 Sanxia Water Strider Virus 3 [SXSSP2554] (ref) AJG39091 Wuhan Mosquito Virus 3 [mosHB230396] (ref) 99 99 AJG39093 Wuhan Mosquito Virus 5 [mosHB231210] (ref) AJG39094 Wuhan Mosquito Virus 6 [mosHB234098] (ref) 99 #AJG39092 Wuhan Mosquito Virus 4 [mosHB231166, etc] (ref) 84 ##Hubei earwig virus 1 [GCM19162, WGML148351] 99 98 Hubei orthomyxo-like virus 1 [SCM47652] AJG39096 Wuhan Mothfly Virus [WHCCII12658] (ref) ACY56282 Quaranfil virus (ref) 100 Quaranjavirus ACY56284 Johnston Atoll virus (ref) 100 Beihai orthomyxo-like virus 1 [BHTSS17872] 92 AJG39087 Shayang Spider Virus 3 [spider138973] (ref) 100 AJG39085 Jiujie Fly Virus [horsefly38597] (ref) Hubei orthomyxo-like virus 2 [QTM27225] 99 AJG39095 Wuhan Mosquito Virus 7 [mosHB235925] (ref) 92 95 AAC25959 Thogoto virus (ref) 100 AED98371 Jos virus (ref) Thogotovirus 100 AAA42968 Dhori virus (ref) Hubei orthomyxo-like virus 3 [ZCM21154] Hubei orthomyxo-like virus 4 [SCM51211] PB1 Phylogeny 100 NP 056657 Influenza B virus (ref) NP 040985 Influenza A virus (ref) 94 Influenza virus 100 AGS48810 Influenza D virus (ref) YP 089653 Influenza C virus (ref) AJG39097 Wuchang Cockroach Virus 2 [ZL8541] ADR77506 Infectious salmon anemia virus (ref) Hubei orthomyxo-like virus 5 [WHBL85127] 88 Beihai orthomyxo-like virus 2 [BHHQ71863] #Changping earthworm virus 2 [CPQY18140] Red: Phylum Arthropoda, Class Insecta EVE vertebrates Highly abundant (> 1% non-rRNA reads) Light blue: Phylum Arthropoda, Subphylum Crustacea EVE arthropods Purple: Phylum Arthropoda, Subphylum Myriapoda EVE nematods Yellow: Phylum Arthropoda, Subphylum Chelicerata Abundant Orange: Phylum Nematoda EVE protists (0.1% ~ 1% non-rRNA reads) Brown: Phylum Annelida EVE flatworms Dark blue: Phylum Mollusca and Sipuncula Black: Subphylum Vertebrata EVE plants Still scratching the surface of virus biodiversity: Grey: other hosts or undertermined EVE fungi may be ~100 million viruses in eukaryotes Diverse, Diverse, Abundant Abundant Chelicerata Annelida & Insecta Crustacea Myriapoda Nematoda Sipuncula Mollusca Platyhelminthes Cnidaria • • Echinodermata ) Tunicata s d a M e 100% M C be confirmed) be highly viruses invertebrate Some type host to clues provides Abundance M r C Y Invertebrate RNA Viruses RNA Invertebrate T G H Q A 90% s p W u M P N l m Retrotransposons C S p i e 80% R r Z S o r c r h X - c I S Q RNA viruses (with RdRp) n i S I n 70% J a M C M L o M C 1 Z d C W 0 n H G 60% H X n S C C F f W C W u S S B W Z o J b S 50% F H L W C T C C B n S I B A M I L S H X abundant (although this needs to to needs this (although abundant o H F C J G H B 40% X i y T S X S l t 9 B W W f X G Z H X H r n 0 e C R X Y X Y X B X B u B 2 W 30% s o C G W X o X X J r Y Z L 0 r X R s C C L X H L N X H X u J Z S e L S N Z p o H H X o Z Z X C X H H S C L S d X H H Z B X W X L T W i C W C H B S C o H x N F Z C C B W m L C J 20% W X Y M i S W i C K W X B B P Z H H H p x F r C R Z H H Z Z 3 H J S Q H H i L p Z C C C H Q S S J M U H J L S S m Z H H S J B J W W Y W B n H i H B P W J L S W S P m H Z H C Y H Y O W y W H ( a S e B B H l C 10% B H M H C Z G C S B Y B B Q H y W F B J i W B S H S W l T l otal otal o 10 s o e p i c 20 e e h t p s 30 n i RNA with with host RNA s s u High abundance viruses r e 40 i i v c (> 0.1% non-rRNA reads) f e 50 o p Low abundance viruses s r e t (< 0.1% non-rRNA reads) s b 60 o m h u r 70 N e p 80 appear to appear rRNA excluded be RdRp Viral methyltransferase Coat protein (S domain) Coat protein (Peptidase A21) Coat protein (Picorna-like) Segmented 10kbp A Helicase 3C-like protease Coat protein (TMV-like) Coat protein (Peptidase A6) Coat protein (CBPV-like) * Viruses Hepe-Virga Tombus-Noda Picorna-Calici Complex Genome * Organizations and * * * Evolution * * * Astro Permutotetra Remarkable Genome Fluidity * * • Frequent recombination, lateral gene * Luteo-Sobemo Narna-Levi Toti-Chryso transfer (inc. with hosts), genome * * reorganization, change in segment * numbers, gene gain and gene loss * * Nido Partit-Picobirna * * RdRp Phylogenies Capsid Phylogenies * New +ve/ds RNA virus (Weivirus) Alvernavirus-like coat protein * Narna-Levi Astro * Viral coat protein Hepe-Virga (Peptidase A21 domain, pfam03566) Mono-Chu, Ophio, Bunya-Arena B and new viruses NG L L G N Viral coat protein NG L G Permutotetra L G * N N: Nucleoprotein (Peptidase A6 domain, pfam01829) N L G: Glycoprotein * GL L N L: Polymerase L L * NGL * L N Luteo-Sobemo 10kbp G N G L * L G N N GG L * L G * N G L Viral coat protein * (S domain, VNN, N L L G * pfam00729) G L L N G L * L G N N L * Tombus-Noda L G LN * L G N L N N * L G N * L G * L G N Orthomyxo L * GL L G N G GG L * L L GG L * * * Recombination of functional modules Novel Mosquito Viruses in Australia LocC Culex australicus 0.05 85 LocA Culex australicus 76 LocA Culex globocoxitus Culex LocD Culex australicus australicus/globocoxitus 95 LocC Culex globocoxitus 100 LocD Culex globocoxitus LocD Culex quinquefasciatus LocA Aedes camptorhynchus LocB Aedes camptorhynchus 100 Aedes camptorhynchus LocD Aedes camptorhynchus LocC Aedes camptorhynchus LocD Aedes alboannulatus Perth LocD: South Guildford LocB: Leschenault Peninsula LocA: Point Douro LocC: Siesta Park 0 100 200km 23 RNA viruses identified, 19 novel Virome Division by Mosquito Genus Mosquito by Division Aedes alboannulatus Aedes camptorhynchus Culex globocoxitus Culex australicus Culex quinquefasciatus A B CDD A C D ACD D Location Keys 12 A: Point Douro s e Low abundance viruses s 10 B: Freshwater larval site u r i High abundance viruses v 8 C: Radio Tower Wetland f o 6 D: Wilkie street r e b 4 m u 2 N 0 r o Toti-Chryso ) 100 Nido j s a A r e N m Reo d Narna R r 80 h l o c / a a s r Partiti i Negev e e v i 60 f l i l o a m t Other negative sense n a o f t o 40 i t f s r o Orthomyxo u o r i p % 20 v o ( r Mononega P 0 Bunya ) y s r e a • • s r u b i r l i 16 v e h A t China) (e.g.
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