Characterisation of a Novel Paramyxovirus Virus from Carp

Home , Aquaparamyxovirus, Feline morbillivirus, Morbillivirus

15-08-2016

Characterisation of a novel Background • Syncytium forming cytopathic effect observed in a variety of cyprinids paramyxovirus virus from carp when undertaking screening, routine diagnostics and reported disease outbreak testing for SVCV and KHV Richard Paley • 22 isolations since 1996 • Limited attempts to characterise generally unsuccessful • Early EM images show possible myxo-like virus 20th Annual Finfish diseases NRL workshop

Growth in cell lines

Group 1 Group 2

Orthomyxovirus Paramyxovirus Enveloped Enveloped 80-120nm ~150nm • Grows in EPC, KF & CCB • Grows in CCB & CHSE Hemagglutinin Hemagglutinin 8 genome segments 1 segment Infectious Salmon Anaemia Virus Pacific Salmon Paramyxovirus • Slow growing >20days • Fast growing <4 days

Classical Genome PCR • High titres , TCID50 ~1x109ml-1 • Low titres, TCID50 • What cell lines • DNA or RNA? • Paramyxovirus ~1x104ml-1 does it replicate • Passage has no affect on in? • Segmented • Orthomyxovirus • Does it have an TCID50 envelope? • Passage reduced TCID50 • Morphology • Can it hemagglutinate?

Log TCID50 Drop with Br-dU treatment Viral genome – RNA

Ability to haemadsorb or haemagglutinate • Test for DNA or RNA • Haemagglutinin protein ISAV • Thymine analogue • Salmon and carp RBCs used inhibits DNA replication • ISAV and PSPV showed haemadsorption and haeamagglutination • Causes a decrease K283 in TCID50 if the virus • All tested isolates were negative has a DNA genome

• RNA genome determined in all

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Presence of an envelope 4 non enveloped isolates were Aquareovirus -

grass carp reovirus DNA G203 J125 • Chloroform test for lipid envelope • Non enveloped isolates gave 1,2,3 L 10kb 11 bands= Aquareovirus • Confirmed by PCR and 4,5,6 M • Causes a drop in sequencing TCID50 if an envelope is • 100% id to Grass carp reovirus (polymerase and capsid present genes) 7,8 S

9 S • Identified at least 2 different viruses 10 S in samples. 11S

Polyacrylamide gel electrophoresis DNA K283 PCR with generic primer sets for Paramyxovirinae and

(PAGE) of viral genome • Primers from Tong et. al (2008) Orthomyxoviridae targeted ≈700bp L polymerase gene for 10kb paramyxovirinae • Remaining enveloped • Consensus/degenerate hybrid primers isolates showed high (iCODEHOP -University of weight smears Washington) for orthomyxo • Primer sets from Dr William Batts USGS Western Fisheries Research Centre - Isolated various • Not resolved by a longer Paramyxoviruses and Orthomyxoviruses from various fish running time, further species (unpublished) phenolic extraction, • Gradient PCR from 42°-52°C G203 F1 G242 F1 M061 F1 native or denaturing PAGE • ISAV and Pacific salmon paramyxovirus as positive controls Test isolates all negative

Group 1 – “myxo”-like TEM

Grp 1- unknown Grp 2 - Reovirus

• myxo-like budding • No budding

• Clear membrane • No lipid membrane

• Size of ~250nm – • Size of ~80nm possibly a bit large for myxoviruses

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Next Generation Sequence analysis Next Generation Sequence analysis • 2 isolates prepared for NGS • Loss of virus during preparation? • Clarified and sucrose purified cell culture harvest

• Double stranded cDNA prepared using tagged random primers and reverse transcriptase then sequenase • Confirmed virus presence by EM negative staining • Roche 454 (1/8th plate per isolate) • 6-12k reads per isolate • Insufficient sequencing depth? • CLC genomics for tag removal and de novo assembly • Offline Blast analysis • Repeated one isolate using HiSeq with DNAse and ribodepletion treatment • Megan, MG RAST, One Codex (Helix I/O) • >10m reads

• No obvious viral sequence, no large contigs • No significant homology • Host genome, ribosomal RNA, micoplasma contamination in one isolate.

Conclusions

• Isolates are a mix of GCRV and a still unknown virus

• Probably not a myxo-like virus or very distantly related

• Possibly a virus family with similar morphology to Para/Orthomyxovirus

Enveloped RNA viruses Iterative Viral Assembler (IVA) pipeline Family/Genus Nucleic acid Configuration Morphology Size (nm) Host

Hypoviridae ds 1 linear Pleomorphic 50-80 Fungi vesicles Cystoviridae ds 3 linear Spherical (icos) 85 Bact • Designed specifically for read pairs Atrerivirus ss 1 + linear Spherical 45-60 Vert sequenced at highly variable depth from Coronaviridae ss 1 + linear Roughly 120 Vert, (fish) Spherical / pleo RNA virus samples Flaviviridae ss 1 + linear Spherical 50 Vert, Insect Togaviridae ss 1 + linear Spherical (icos) 65-70 Vert, Insect, (fish) • Shown to produce significantly higher Paramyxoviridae ss 1 – linear Spherical 150 Vert, (fish) quality assemblies than existing Arenaviridae ss 2 – linear Spherical 60-300 Vert approaches Orthomyxoviridae ss 8 – linear Spherical 80-120 Vert, (fish)

Bunyaviridae ss 4-5 +/- linear Spherical / pleo 80-120 Vert, Insect, Plant Retroviridae ss dimer 1 + linear Spherical 80-100 Vert (fish) • M061 de novo assembly  single contig of 16,807bp

Hunt et al 2015 Bioinformatics 31(14):2374-6

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Mapping of reads to the novel virus genome (HiSeq dataset) Mapping of reads to the novel virus genome (MiSeq dataset)

Trimmed paired Trimmed paired Raw reads reads Unpaired R1 Unpaired R2 Mapped reads Percentage Coverage Raw reads reads (w/o carp) Unpaired R1 Unpaired R2 Mapped reads Percentage Coverage Sample 16 52,070 48,994 1,820 130 0 0% - M061 10,871,535 6,566,088 78,141 7,071 237,895 1.78% 1854 +/- 761 Sample 17 90,779 86,407 2,294 129 345 0.2% 1.97 +/- 1.98 Sample 18 96,125 92,985 1,617 79 8 0% -

RNA-directed RNA polymerase L, partial [Human parainfluenza virus 3] Sequence ID: gb|AHX22063.1|Length: 2233Number of Matches: 1

Score Expect Method Identities Positives Gaps 838 bits(2166) 0.0 Compositional matrix adjust 560/1746(32%) 879/1746(50%) 134/1746(7%)

Query 224 YPPVEEHEMLGLLVTPVYVLIHMGENLIS--LSPEQVLMICDVVEGRLRIDCVLTISGEE 281 Y +E+ + L LL+ P VLI +N ++PE VLM CDV+EGR I + Sbjct 201 YNLLEDQKNL-LLIHPELVLILDKQNYNGYLITPELVLMYCDVIEGRWNISACAKLD--- 256

Query 282 TLPTVSSF----RAVLQHLDSLFPVMGNSMYDVQKLIEALVQGLILEFDPENPSNSSFLS 337 P + S + + +D LFP+MG +DV L+E L LI DP +FL+ Sbjct 257 --PKLQSMYQKGNNLWEVIDKLFPIMGEKTFDVISLLEPLALSLIQTHDPVKQLRGAFLN 314

Query 338 FVTDEFV------KALTPFLSEPVKWTFKLVELLDCDTPDKLAEWMCLYRLCGHPHLCAY 391 V E +++ FLS V + K++++ D T D++AE +R GHP L A Sbjct 315 HVLSEMELIFESRESIKEFLS--VDYIDKILDIFDKSTIDEMAEIFSFFRTFGHPPLEAS 372

Query 392 KAAAKVRKHIKGEKILDLTDIHKTYASFCQIYITGYVHKHGGMWPAMEIDERHANWLFKK 451 AA KVRK++ EK L I+K +A FC I I GY +HGG WP + + + ++ Sbjct 373 IAAEKVRKYMYIEKQLKFDTINKCHAIFCTIIINGYRERHGGQWPPVTLPDHAHEFIINA 432

Query 452 LKSGECPTEAEALQHYRDFLYIDWGKSQDVNLEEDLSMFMKDKAISPPMSHWDCVFSKSL 511 S + A+ +Y+ F+ I + K + L+EDL+++MKDKA+SP S+WD V+ S Sbjct 433 YGSNSAISYENAVDYYQSFIGIKFNKFIEPQLDEDLTIYMKDKALSPKKSNWDTVYPASN 492

Query 512 VSYDVPWYNGSRRLVQTVLNSEKMEPQHYLDYVTSGDYLKDDDYCMSYSLKEREVNTDGR 571 + Y N SRRLV+ + K +P LDYV SGD+L D ++ +SYSLKE+E+ +GR Sbjct 493 LLYRTNASNESRRLVEVFIADSKFDPHQILDYVESGDWLDDPEFNISYSLKEKEIKQEGR 552

Query 572 IFGKMTIDMRGAQVLGEALLAHGIAKYFPDNGMVKGEIDLAKNLFHLAATSQGSFRG-KN 630 +F KMT MR QVL E LLA+ I K+F +NGMVKGEI+L K L ++ + + N Sbjct 553 LFAKMTYKMRATQVLSETLLANNIGKFFQENGMVKGEIELLKRLTTISISGVPRYNEVYN 612

Query 631 TSHSDQDDEPGGN------EYQATVI------SGSC--TTDIQKYCLNWR 666 S S DD N E+++T I + SC TTD++KYCLNWR TAGCACCCA – putative transcriptional start Sbjct 613 NSKSHTDDLKTYNKISNLNLSSNQKSKKFEFKSTDIYNDGYETVSCFLTTDLKKYCLNWR 672

Query 667 EESVDLFAKALDKIYGLDNFFNWHCKRQKLSIPYVADPFCVP------LFDKRPKKPPLD 720 ACAAAAA – putative poly A initiation site ES LF + ++I+GL+ FNW R + S YV DP+C P L + P D Sbjct 673 YESTALFGETCNQIFGLNKLFNWLHPRLEGSTIYVGDPYCPPSDKEHILLEDHP-----D 727

Respirovirus Aquaparamyxovirus Paramyxoviridae

(proposed) Fer-de-Lance paramyxovirus NJ Canine distemper virus Dolphin morbillivirus Rinderpest virus Morbillivirus Carp paramyxovirus Human parainfluenza virus 1 Rubulavirus Measles virus Feline morbillivirus

Salem virus Beilong virus Sendai virus Human parainfluenza virus 3 Nipah virus J-virus Henipavirus Hendra virus Porcine rubulavirus Tupaia paramyxovirus

Bovine parainfluenza virus 3 virus parainfluenza Bovine Mossman virus Human parainfluenza virus 2 Cedar virus Parainfluenza virus 5

Mumps virus Nariva virus Pacific salmon paramyxovirus salmon Pacific Mojiang virus Menangle virus Salem virus Mojiangparamyxovirus salmon Atlantic virus Cedar virus Tioman virus Hendra virus Henipavirus Nariva virus Nipah virus Fer-de-Lance paramyxovirus Mossman virus Avian paramyxovirus 3 Avian paramyxovirus 4 Atlantic salmon paramyxovirus Tupaia paramyxovirus Pacific salmon paramyxovirus Aquaparamyxovirus J-virus AvianAvian paramyxovirus paramyxovirus 8 7 Avian paramyxovirus 2 Bovine parainfluenza virus 3 (proposed)

Avian paramyxovirus 6 Human parainfluenza virus 3 Beilong virus Avian paramyxovirus 5 Sendai virus Respirovirus Human parainfluenza virus 1 Feline morbillivirus Avian paramyxovirus 12 Measles virus Carp paramyxovirus

Parainfluenza virus 5 Rinderpest virus Rinderpest Human parainfluenza virus 2

Avian paramyxovirus 9 Mumps virus Rubulavirus Newcastledisease virus Dolphin morbillivirus Dolphin Porcine rubulavirus

Canine distemper virus distemper Canine Avulavirus Menangle virus Pigeon paramyxovirus 1 paramyxovirus Pigeon Tioman virus Morbillivirus Avian paramyxovirus 7 Avian paramyxovirus 3

Avian metapneumovirus Avian paramyxovirus 4 Newcastle disease virus

Human metapneumovirus Pigeon paramyxovirus 1 Avian paramyxovirus 9 Avulavirus Murine pneumonia virus pneumonia Murine Avian paramyxovirus 12 Avian paramyxovirus 2 Avian paramyxovirus 8

Human respiratory syncytial virus syncytial respiratory Human Avian paramyxovirus 5 Bovine respiratory syncytial virus syncytial respiratory Bovine Avian paramyxovirus 6 Avian metapneumovirus Metapneumovirus Human metapneumovirus Murine pneumonia virus Metapneumovirus Human respiratory syncytial virus Pneumovirus Bovine respiratory syncytial virus 0.2 Ebola virus Pneumovirus

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Paramyxoviridae

VIRION - Enveloped, spherical. Diameter of about 150 nm.

GENOME - Negative-stranded RNA linear genome, about 15 kb in size. Encodes for eight proteins.

TAGCACCCA – putative transcriptional start ACAAAAA – putative poly A initiation site

Isolated Aquaparamyxovirus Isolate Host Origin Reported diaease with VIRION - Enveloped, spherical. Diameter of about 150 nm. G106 Grass, silver black, bighead carp Israel Mortalities - GENOME- Negative-stranded RNA linear genome, about 17 kb in size. Encodes for nine proteins. F201 mirror/crucian hybrids - Disease outbreak -

G200 Ghost koi - NONE - G203 Golden Tench Israel NONE - G242 KOI UK Poss bacterial ulcers - H205 Common Carp - NONE - Fungal H279 Koi UK Lesion behind eye/ infection J107 Common Carp - NONE - J120 Common Carp UK Mortalities CyHV-1 J125 Ghost koi - NONE - J363 Common Carp UK NONE - Slender, 20% enlarged spleen grey fungal K246 koi/goldfish UK covering on 50% of fish infection K283 mirror - NONE - K288 koi - NONE - MO44 Common Carp - mortalities Parasites MO54 Common Carp - Similar pathology to Carp pox observed - MO61 Common Carp - NONE - fungal PO31 Roach/common carp - Sunken eyes and fin rot infection PO54 Koi - Mortalities - 45IT - - - - 966 - - - - 885 - - - -

Challenge Summary • Finally have identification ……..new virus within the • IP/cohab in both carp Cumulative mortality over 30 days at 17ºC paramyxoviridae - species/genus. and tench Isolate Days post infection 3 6 9 12 15 18 21 24 27 30 • Characterisation ongoing - genotypic and phenotypic. • 30 fish of each group 451 1 1 • Likely to promote reassessment of the proposed 545 5 7 7 Aquaparamyxovirus - cypriniparamyxovirus. • 0.1 ml clarified cell 625 2 5 5 culture harvest (approx. 669 1 4 8 9 • Importance of purity of sample, library prep and depth 3.5 TCID /fish) 50 885 3 3 of sequencing. 960 2 6 11 13

• Mortalities in IP injected control 1 1 1 1 1 1 1 1 1 1 • Importance of reanalysing data sets and keeping carp only, but up to 43% finger on the pulse of new bioinformatics tools.

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58 Canine distemper virus 58 Canine distemper virus 94 Dolphin morbillivirus Dolphin morbillivirus Rinderpest virus NJ 94 99 Rinderpest virus 87 Measles virus 99 Feline morbillivirus 87 Measles virus Acknowledgements 40 Feline morbillivirus 100 Beilong virus 40 J-virus 100 Beilong virus Tupaia paramyxovirus J-virus Connor Sharp 48 67 67 Tupaia paramyxovirus 92 Mossman virus 48 Mossman virus Annabel Rice 93 Nariva virus 92 96 Mojiang virus 93 Nariva virus 96 Kelly Bateman – EM 34 Salem virus Mojiang virus 34 Salem virus 36 Cedar virus Irene Cano Cejas – NGS virus prep Cedar virus 94 Hendra virus 36

100 Nipah virus 94 Hendra virus Ronnie van Aerle - Bioinformatics 99 Fer-de-Lance paramyxovirus 100 Nipah virus David Stone – molecular virology & bioinformatics 100 Atlantic salmon paramyxovirus 99 Fer-de-Lance paramyxovirus Pacific salmon paramyxovirus 100 Atlantic salmon paramyxovirus

100 Bovine parainfluenza virus 3 Pacific salmon paramyxovirus Bill Batts (USGS WFRC) – primer sets & PSPV 53 82 Human parainfluenza virus 3 100 Bovine parainfluenza virus 3 53 82 Knut Falk (NVI) – ASPV 97 Sendai virus Human parainfluenza virus 3 100 Human parainfluenza virus 1 97 Sendai virus Defra funding Carp paramyxovirus 100 Human parainfluenza virus 1 Parainfluenza virus 5 66 98 Carp paramyxovirus 74 Human parainfluenza virus 2 98 Parainfluenza virus 5 81 66 Mumps virus 74 Human parainfluenza virus 2 97 Porcine rubulavirus 81 Mumps virus Fer-de-Lance paramyxovirus Menangle virus

Carp paramyxovirus 97 Human parainfluenza virus 1 Porcine rubulavirus

Salem virus Sendai virus Human parainfluenza virus 3 Nipah virus 99 Tioman virus Hendra virus Porcine rubulavirus Menangle virus

Bovine parainfluenza virus 3 virus parainfluenza Bovine Human parainfluenza virus 2 Cedar virus Parainfluenza virus 5 Mumps virus

Pacific salmon paramyxovirus salmon Pacific Avian paramyxovirus 7 Menangle virus 63

Atlantic salmon paramyxovirus salmon Atlantic 99 Tioman virus Mojiang virus Tioman virus Nariva virus 90 Avian paramyxovirus 3 Avian paramyxovirus 3 Avian paramyxovirus 3 Mossman virus Avian paramyxovirus 4 90 Tupaia paramyxovirus 63 J-virus AvianAvian paramyxovirus paramyxovirus 8 7 Avian paramyxovirus 2 Avian paramyxovirus 4 Avian paramyxovirus 6 Beilong virus Avian paramyxovirus 4 Avian paramyxovirus 5 73 Feline morbillivirus Avian paramyxovirus 12 Measles virus Newcastle disease virus Rinderpest virus Rinderpest 100 Avian paramyxovirus 7

Avian paramyxovirus 9

Newcastledisease virus

Dolphin morbillivirus Dolphin 98 Canine distemper virus distemper Canine Pigeon paramyxovirus 1 Pigeon paramyxovirus 1 paramyxovirus Pigeon 73 Newcastle disease virus 16 98 100

Avian metapneumovirus Avian paramyxovirus 9

Human metapneumovirus 98 Pigeon paramyxovirus 1

Murine pneumonia virus pneumonia Murine Avian paramyxovirus 12

33 98 Avian paramyxovirus 9 Human respiratory syncytial virus syncytial respiratory Human

Bovine respiratory syncytial virus syncytial respiratory Bovine 97 Avian paramyxovirus 2 34 Avian paramyxovirus 12 Avian paramyxovirus 8 0.2 97 Avian paramyxovirus 2 31 Avian paramyxovirus 5 34 Avian paramyxovirus 8 34 Avian paramyxovirus 6 31 Avian paramyxovirus 5 96 Avian metapneumovirus 34 Avian paramyxovirus 6 Human metapneumovirus 96 Avian metapneumovirus 100 Murine pneumonia virus Human metapneumovirus 97 Human respiratory syncytial virus 100 Murine pneumonia virus 100 Bovine respiratory syncytial virus Human respiratory syncytial virus Ebola virus 97 100 Bovine respiratory syncytial virus

0.2 Ebola virus

Bovine respiratory syncytial virus Human respiratory syncytial virus NJ High-throughput sequencing runs on different platforms

Murine pneumonia virus Human metapneumovirus Avian paramyxovirus 5

Avian paramyxovirus 4 paramyxovirus Avian Avian paramyxovirus 6 Avian paramyxovirus 9

AvianPigeon paramyxovirus paramyxovirus 1 12 Raw reads No of contigs Min contig length Max contig length Mean contig length Newcastle disease virus disease Newcastle Avian paramyxovirus 8 Avian paramyxovirus 2 Avian metapneumovirus

Avian paramyxovirus 3 paramyxovirus Avian EDGARJ363 6,379 48 106 791 364 100

Human parainfluenza virus 1 EDGARK283 12,707 503 100 2,934 851 Avian paramyxovirus 7 paramyxovirus Avian

Carp paramyxovirus 98 Ebola virus 454

98 97

Tioman virus Tioman 96 90 100 100

99 Menangle virus Menangle 3431 Trimmed paired 34 73

98 16 Raw reads reads (w/o carp) Unpaired R1 Unpaired R2 Mapped reads Percentage Coverage

74 81

63 Porcine rubulavirus Porcine 66 Human parainfluenza virus 3 M061 10,871,535 6,566,088 78,141 7,071 237,895 1.78% 1854 +/- 761

Sendaivirus virusMumps 53 HiSeq

100 82 36

Bovine parainfluenza virus 3 97 99

48 9994 40 58

34 Trimmed paired

100 100 94 67

87 100 Pacific salmon paramyxovirus 92

Parainfluenza virus 5 virus Parainfluenza Raw reads reads Unpaired R1 Unpaired R2 Mapped reads Percentage Coverage 93

100 virus distemper Canine Sample M061 52,070 48,994 1,820 130 0 0% - Human parainfluenza virus 2 virus parainfluenza Human Sample M054 90,779 86,407 2,294 129 345 0.2% 1.97 +/- 1.98

Atlantic salmon paramyxovirus

Nipah virus J-virus MiSeq Dolphin morbillivirus Dolphin Sample P031 96,125 92,985 1,617 79 8 0% -

0.2

Fer-de-Lance paramyxovirus virus Measles

Rinderpest virus Rinderpest

Hendra virus virus Beilong Nariva virus Nariva

Cedar virus virus Mojiang

Feline morbillivirus Feline Mossman virus Mossman

Salem virus Tupaia paramyxovirus Tupaia