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• (van Hulten et al., 2001; Yang et al., 2001)

Crustacean Genome Exploration WSSV: enigmatic Envelope Nucleocapsid Embedded with structural proteins responsible shell containing Reveals the Evolutionary Origin for host cell entry genomic DNA of Deadly Shrimp Virus

Satoshi Kawato, Hidehiro Kondo, and Ikuo Hirono Tail-like extension Tokyo University of Marine Science and Technology, Japan Envelope extension 250-380 nm of unknown function

Inouye et al. (1994) • Double-stranded DNA virus (circular, ca. 300 kbp)

• Extremely broad host range (Lo et al., 1996; Otta et al., 1999) • Few relatives reported: isolated taxonomic position (family Nimaviridae)

PAG XXVII • Little knowledge on evolution and phylogeny January 12, 2019

Studying shrimp (and their pathogens) does matter Viral “fossils” buried in the host genome

shrimp aquaculture production (tons) 2016 fishery commodities global export Marsupenaeus japonicus BAC clone Mj024A04 (Koyama et al., 2010) 6,000,000 (value, USD)

2016 production: 5,000,000 $22.9 billion 5.18 million tons (16%) 4,000,000

3,000,000

2,000,000 (modified from Koyama et al., 2010)

1,000,000 • WSSV homologs in localized in the cell nucleus as repetitive

elements (Koyama et al., 2010) 0 Grand total: $142.8 billion 1980 1990 2000 2010 • Over 30 WSSV homologs identified in M. japonicus genome

(Shitara, unpublished; Wang, unpublished)

Whiteleg shrimp Asian tiger shrimp Kuruma shrimp Litopenaeus vannamei Penaeus monodon Marsupenaeus japonicus 6

Shrimp aquaculture production

White spot disease (WSD) Viral "fossils" buried in the host genome

Nimavirus WSD outbreak History (tons)

1992~: First outbreak in Asia 3000 Viral DNA Viral DNA integrated into the shrimp genome ~2000: $7 to $8B estimated loss 2000 (Asia and the Americas) 1000 ~2010s: Spread throughout the world 0 Symptoms 1960 1970 1980 1990 2000 2010 (year) Countries/regions affected by WSD • White spots on the exoskeleton Shrimp chromosomes Shrimp chromosomes • Body discoloration containing viral DNA • Lethargy • genomes harbor footprints of ancient WSSV • 100% mortality within 3-10 days relatives (nimaviruses) (Koyama et al., 2010, Huang et al., 2011; Rosenberg et al., 2015)

Causative agent Objective virus (WSSV) Comparative genomic analysis of (fossilized) nimavirues to elucidate the evolutionary history of WSSV

Objective Comparative genomic analysis of fossilized WSSV relatives to elucidate the evolutionary history of WSSV

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Methods Summary Comparative genomic analysis of Nimaviridae

XX% identity

Crustacean samples Genome survey De novo assembly Homology search (14 species) sequencing (CLC Genomics Workbench) (BLAST/HMMER/HHpred) (Illumina MiSeq)

Genes inherited Genes acquired WSSV-specific Comparative from the common during evolution genes Ancestral genes ancestor Genes acquired WSSV-specific Core genes subsequently lost genomic analyses during evolution genes in some nimaviruses Distribution of WSSV orthologs in Nimaviridae Predicted genes in the WSSV CN01 genome: 177 Nimaviral core genes: 28 WSSV-specific genes: 92

Nimaviral sequences in decapod Many WSSV characteristic genes are young genes

Kuruma shrimp Sakura shrimp Metapenaeus ensis Sergia lucens

Western king prawn Yamato shrimp WSS_VP family: the most Melicertus latisulcatus multidentata abundant WSSV structural proteins

Asian tiger shrimp Pacific grass shrimp Penaeus monodon Palaemon pacificus VP28 VP24 Banana shrimp Brush clawed shore crab Outside Fenneropenaeus merguiensis Hemigrapsus takanoi (envelope)

Pacific white shrimp Green crab ◀VP28 Inside Litopenaeus vannamei Carcinus sp. ◀VP26 VP26 (tegument) ◀VP24 Greezyback shrimp Metapenaeus ensis Sesarmops intermedium

Shiba shrimp Metapenaeus joyneri Chiromantes dehaani

Nimaviral sequences detected in 12/14 species (modified from van Hulten et al., 2000)

Diversity of WSSV relatives Many WSSV characteristic genes are young genes

▲VP24 evolved from VP28

Blue: nimavirus genomes identified in this study Green: nimaviruses isolated as viral particles * Fossilized nimavirus in a sesarmid crab genome

(Rosenberg et al., 2015) *

• WSSV relatives of different “genera”  Diversity and long history of Nimaviridae ▲VP24 is unique to WSSV ▲WSS_VP arose recently in history

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Expansion of host-viral interface by unique accessory Is WSSV a distant offshoot of baculo-like ? gene duplications

Outer circle Double-stranded DNA viruses infecting WSSV-specific genes WSSV-specific genes Nucleocytoplasmic Nuclear -specific absent in some isolates Repetitive regions large DNA viruses large DNA viruses (NCLDV) (Iyer et al., 2001 ) (NALDVs) Inner circle (Wang et al., 2011) Envelope proteins

Putative transcription factors and ubiquitin ligases

Single-copy genes

Distribution of WSSV-specific paralogs in the WSSV CN01 genome Branching ribbons denote the phylogenetic relationships among paralogs.

• Unique paralogues accounting for 48% (42/92) of WSSV-specific genes Per conserved among insect DNA viruses, which are required for viral • WSSV-specific genes: associated with host-viral interactions Nimaviridae • Lacking in some isolates: non-essential accessory genes entry into host midgut cells

Left: model of Right: PIFs in the

Insect dsDNA virus protein homologs in Nimaviridae Is WSSV a distant offshoot of baculo-like viruses?

PIF0 PIF1 PIF2 PIF5

PIF3

Left: model of baculovirus infection (Slack and Arif, 2006) Right: PIFs in the baculovirus envelope

Per os infectivity factors (PIFs): a set of envelope proteins conserved among insect DNA viruses, which are required for viral entry into host midgut cells Maximum likelihood phylogenetic tree of arthropod dsDNA viruses based on seven NALDV core genes

Origin of Nimaviridae

Insect dsDNA virus protein homologs in Nimaviridae Summary

Ancestral Function Name Baculoviridae Nudiviridae Hytrosaviridae WSSV Nimaviridae Transcription (6) p47 ● ● – – – lef-8 ● ● ● – – lef-9 ● ● ●  Host genome survey revealed previously vlf-1 ● ● – – – lef-4 ● ● ● – – undetected diversity of WSSV relatives lef-5 ● ● ● – – Replication (2) DNA polymerase ● ● ● ● ● Helicase ● ● ● – – Structural proteins (12)  Gene duplications enriched WSSV-specific Oral infectivity (6) p74 (PIF-0) ● ● ● ● ● pif-1 ● ● ● ● ● genes involved in host-viral interactions pif-2 ● ● ● ● ● pif-3 ● ● ● ● ● pif-4/19kda ● ● – – – pif-5/odv-e56 ● ● ● ● ●  Insect dsDNA virus homologs in the ancestral Morphogenesis (2) ac68 ● ● – – – 38K ● ● – – – nimavirus gene set suggest a shared Unknown (2) vp91/p95 ● ● – – – vp39 ● ● – – – phylogentetic origin of arthropod dsDNA Others (2) ac81 ● ● ● – – p33 ● ● ● ● ● Number of NALDV viruses core gene homologues 20 20 14 7 7 ●Newly identified in this study ( Modified from Wang et al., 2011)

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Half of the conserved genes are of unknown function Acknowledgement Nimaviral core genes (28) • Jumroensri Thawonsuwan Function in WSSV WSSV ORF • Hiroyuki Yokooka Structural proteins (11) Envelope proteins (2) wsv293a, wsv327 wsv037, wsv220, wsv271, wsv289, proteins (7) wsv332, wsv360, wsv415 Unknown (2) wsv131, wsv161 Non-structural proteins (17) DNA polymerase wsv514 Putative TATA binding protein wsv303 Putative protein kinase wsv423 Reference Latency-related gene wsv427 Kawato, S., Shitara, A., Wang, Y., Nozaki, R., Kondo, H., & Hirono, I. (2018). wsv026, wsv133, wsv137, wsv139, Crustacean Genome Exploration Reveals the Evolutionary Origin of White Spot wsv192, wsv267, wsv282, Hypothetical protein (13) Syndrome Virus. Journal of virology, JVI-01144. wsv285, wsv313, wsv343, wsv433, wsv440, wsv447 Thank you

Marsupenaeus japonicus endogenous nimavirus Nuclear arthropod-specific large DNA viruses (NALDV)

Baculoviridae Nudiviridae Hytrosaviridae WSSV

• Rod-shaped, enveloped virions • Circular genomes interspersed with repeat regions • ~220 kbp genome, 2 fragments • Replication in the host cell nucleus • At least 39 WSSV orthologs • NALDV core genes • Most distantly related to WSSV (Wang et al., 2011) • Homologs present in other 5 species

Penaeus monodon endogenous nimavirus

• ~200 kbp genome, 3 fragments • At least 39 WSSV orthologs • Homologs present in 2 other species

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