Transcriptomic profiles of Senegalese aole infected with nervous necrosis virus reassortants presenting different degree of virulence

July 2018 | Volume 9 | A icle 16261
O iginal esea ch
published: 17 July 2018
doi: 10.3389/ immu.2018.01626
F on ie s in Immunology | www. on ie sin.o g
Edi ed by:
Lluis To ,
Au onomous Uni e si y o
Ba celona, Spain
Re iewed by:
Wenbin Zhan,
Ocean Uni e si y o China, China
Giuseppe Scapiglia i,
Uni e si à degli S udi della
Tuscia, I aly
*Co espondence:
Juan J. Bo ego
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Compa a i e Immunology,
a sec ion o he jou nal
F on ie s in Immunology
Recei ed: 22Feb ua y2018
Accep ed: 02July2018
Published: 17July2018
Ci a ion:
LabellaAM, Ga cia-RosadoE,
BandínI, DopazoCP, Cas oD,
AlonsoMC and Bo egoJJ (2018)
T ansc ip omic P o iles o Senegalese
Sole In ec ed Wi h Ne ous Nec osis
Vi us Reasso an s P esen ing
Di e en Deg ee o Vi ulence.
F on . Immunol. 9:1626.
doi: 10.3389/ immu.2018.01626
T ansc ip omic P o iles o
senegalese sole in ec ed Wi h
ne ous nec osis Vi us
easso an s P esen ing
Di e en Deg ee o Vi ulence
Alejand o M. Labella1, Es he Ga cia-Rosado1, Isabel Bandín2, Ca los P. Dopazo2,
Dolo es Cas o1, M. Ca men Alonso1 and Juan J. Bo ego1*
1 Depa amen o de Mic obiología, Facul ad de Ciencias, Uni e sidad de Malaga, Malaga, Spain, 2 Depa amen o
de Mic obiología y Pa asi ología, Ins i u o de Acuicul u a, Uni e sidad de San iago de Compos ela, San iago de Compos ela,
Spain
Be anoda i uses [ne ous nec osis i us (NNV)] a e he causa i e agen o he i al encepha-
lopa hy and e inopa hy, a disease ha a ec s cul u ed Senegalese sole (Solea senegalensis).
NNV easso an s, combining genomic segmen s om edspo ed g oupe ne ous nec osis
i us (RGNNV) and s iped jack ne ous nec osis i us (SJNNV) geno ypes, ha e been p e i-
ously isola ed om se e al ish species. The wild- ype easso an wSs160.03, isola ed om
Senegalese sole, has been p o en o be mo e i ulen o sole han he pa en al geno ypes
(RGNNV and SJNNV), causing 100% mo ali y. Mu a ions a amino acids 247 (se ine o
alanine) and 270 (se ine o aspa agine) in he wSs160.03 capsid p o ein ha e allowed us o
ob ain a mu an easso an ( Ss160.03247+270), which p o okes a 40% mo ali y dec ease.
In his s udy, he RNA-Seq echnology has been used o compa a i ely analyze Senegalese
sole ansc ip omes in wo o gans (head kidney and eye/b ain) a e in ec ion wi h wild- ype
and mu an s ains. A o al o 633 genes we e di e en ially exp essed (DEGs) in animals
in ec ed wi h he wild- ype isola e (wi h highe i ulence), whe eas 393 genes we e di e en-
ially exp essed in animals in ec ed wi h he mu an s ain (37.9% dec ease in he numbe o
DEGs). To s udy he biological unc ions o de ec ed DEGs in ol ed in NNV in ec ion, a gene
on ology (GO) en ichmen analysis was pe o med. Di e en GO p o iles we e ob ained in he
ollowing subclasses: (i) biological p ocess; (ii) cellula componen ; and (iii) molecula unc ion,
o each i al s ain es ed. Immune esponse and p o eolysis ha e been he p edominan
biological p ocess a e he in ec ion wi h he wild- ype isola e, whe eas he in ec ion wi h he
mu an s ain induces p o eolysis in head kidney and inhibi ion o asculogenesis in ne ous
issue. Rega ding he immune esponse, genes coding o p o eins ac ing as media o s
o ype I IFN exp ession (DHX58, IRF3, IRF7) and IFN-s imula ed genes (ISG15, Mx, PKR,
Gig1, ISG12, IFI44, IFIT-1, o name a ew) we e up egula ed in animals in ec ed wi h he wild-
ype isola e, whe eas no-di e en ial exp ession o hese genes was obse ed in samples
inocula ed wi h he mu an s ain. The di e en ansc ip omic p o iles ob ained could help o
be e unde s and he NNV pa hogenesis in Senegalese sole, se ing up he impo ance as
i ulence de e minan s o amino acids a posi ions 247 and 270 wi hin he RNA2 segmen .
Keywo ds: Solea senegalensis, easso an ne ous nec osis i us, ansc ip ome, na-seq, di e en ially
exp essed genes
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Labella e al. T ansc ip omic Analysis o NNV-In ec ed Sole
F on ie s in Immunology | www. on ie sin.o g July 2018 | Volume 9 | A icle 1626
inT ODUcTiOn
Senegalese sole (Solea senegalensis, Kaup 1858), a la ish belonging
o he Soleidae amily, p esen s a high impo ance in he Eu opean
aquacul u e due o i s as g ow h a e in cap i i y and o i s high
comme cial alue (1). Nowadays, he main limi ing ac o o
Senegalese sole cul u e is he appea ance o mic obial epizoo ic
ou b eaks, being he i al encephalopa hy and e inopa hy (VER)
o i al ne ous nec osis one o he mos impo an , since disease
ou b eaks achie e almos 100% o mo ali y in cul u es o sole (2).
Ne ous nec osis i us (NNV) is he e iological agen o VER,
a ec ing mo e han 40 ma ine and eshwa e ish species wo ld-
wide (2, 3). NNV (genus Be anoda i us, amily Noda i idae)
is a non-en eloped icosahed al i us wi h wo single-s anded
posi i e-sense RNA segmen s (RNA1, 3.1 Kb, encodes he RNA-
dependen RNA polyme ase; RNA2, 1.4 Kb, encodes he capsid
p o ein) (4, 5). A subgenomic segmen [RNA3, 0.4kb, encoding
he non-s uc u al p o eins B1 and B2, has been desc ibed by
se e al au ho s (6–8)]. Based on he a iable T4 egion (RNA2),
NNV has been classi ied in o ou species: s iped jack ne ous
nec osis i us (SJNNV), ige pu e ne ous nec osis i us,
edspo ed g oupe ne ous nec osis i us (RGNNV), and ba in
lounde ne ous nec osis i us (BFNNV) (3). Mo eo e , se e al
RGNNV-SJNNV easso an isola es, p esen ing bo h genomic
segmen combina ions, SJNNV/RGNNV and RGNNV/SJNNV,
ha e been isola ed om se e al ish species (9–11). A highly
i ulen RGNNV/SJNNV easso an , ha bo ing a RGNNV- ype
RNA1 and SJNNV- ype RNA2 segmen s, has been isola ed om
Senegalese sole, and named wild- ype (w ) isola e (wSs160.03)
(10). Compa ed o SJNNV geno ype, his i al easso an isola e
displays sequence di e ences a ec ing amino acidic posi ions
247 and 270 a he C- e minal ex eme o he capsid p o ein,
which seems o be ela ed o he inc ease o i s i ulence
(12, 13). In ac , a mu an o his i al easso an ob ained by
e e se gene ic, p esen ing subs i u ions o se ine o alanine a
amino acid posi ion 247 and o se ine o aspa agine a esidue
270 (named Ss160.03247+270), causes a educ ion in sole mo ali y
o 40% compa ed o he w isola e (14).
RNA-Seq echnology is a sequencing-based me hod ha
allows o s udy he en i e ansc ip ome in a high- h oughpu and
quan i a i e manne , becoming he p ima y echnology o be used
o gene exp ession p o iling (15). One o i s majo ad an ages is
ha i can cap u e ansc ip ome dynamics ac oss di e en is-
sues o condi ions wi hou sophis ica ed no maliza ion o da a
se s (16). The e o e, his echnology p o ides aluable da a o
unde s anding i us–hos in e ac ions, and i has been used o
s udy pa hogenic p ocesses du ing ish i us in ec ion (17–21).
This in o ma ion is essen ial o unde s and ish immuni y agains
mic obial pa hogens, and o de elop e ec i e s a egies o p e en
ish diseases.
The aim o he p esen s udy is o iden i y di e en ially exp essed
genes (DEGs) in ol ed in Senegalese sole esponse agains in ec-
ions wi h NNV easso an s wi h di e en deg ees o i ulence.
RNA-Seq echnology has been used o ob ain he ansc ip omic
p o iles om Senegalese sole head kidney (one o he majo
lymphohaema opoie ic o gans in ish) and ne ous issues (pools
o eye and b ain, he NNV a ge o gans) a e in ec ion wi h
he w isola e wSs160.03, and he mu an s ain Ss160.03247+270.
Di e en ypes o accines ha e been es ed in a ious ish spe-
cies ele an o he aquacul u e sec o (22). Howe e , no accine
o Senegalese sole agains NNV has been epo ed. The esul s
ob ained p o ided ele an in o ma ion abou sole–NNV in e -
ac ion and, he e o e, o he de elopmen o new s a egies o
con ol his i al disease.
MaTe ials anD MeThODs
Vi us and cell cul u e
The be anoda i uses used in his s udy we e he w isola e
(wSs160.03) and he mu an s ain ha bo ing pinpoin mu a ions
a posi ion 247 and 270 o he capsid p o ein ( Ss160.03247+270)
(10, 14). Bo h i al s ains p esen a combina ion o segmen s
RGNNV (RNA1)/SJNNV (RNA2). The GenBank accession
numbe s o wSs160.03 (SpSs-IAusc160.03) genome segmen s
a e FJ803911 (RNA1) and FJ803923 (RNA2). Vi uses we e p o-
paga ed on E-11 cells (23) in Leibo i z medium (L-15, Gibco,
Paisley, UK) supplemen ed wi h 2% e al bo ine se um (FBS,
Gibco) and 0.5% penicillin/s ep omycin (Sigma-Ald ich,
S . Louis, MO, USA). Inocula ed cells we e incuba ed a 25°C o
up o 15days. Cell cul u e supe na an s we e eco e ed a e he
appea ance o cy opa hic e ec s, and cla i ied by cen i uga ion
a 5,000×g o 10min a 4°C. Vi al suspensions we e s o ed a
−80°C. Vi al i a ions we e pe o med in 96-well pla es (Nunc,
The mo Elec on LED GmbH, Wiesbaden, Ge many) using he
50% issue cul u e in ec ious dose (TCID50) me hod (24).
expe imen al in ec ions and sampling
P ocedu es
Th ee g oups o 30 Senegalese sole ju enile specimens (5–10g)
we e acclima ized o one week a he aqua ium se ices o he
Uni e si y o Malaga (Spain). Animals we e ed wi h a com-
me cial die (Bioma , Palencia, Spain), and main ained in 100-L
aqua ia wi h ae a ion unde s able empe a u e (18±0.5°C) and
salini y (33–35g/L). Fish used in his s udy ha e been ea ed
acco ding o he Guidelines o he Eu opean Union Council
(Di ec i e 2010/63/EU) and he Spanish di ec i e (RD 53/2013).
Th ee expe imen al g oups we e conside ed: (A) nega i e con-
ol, injec ed wi h L-15 Leibo i z medium; (B) animals injec ed
wi h he w isola e, wSs160.03; and (C) animals injec ed wi h he
mu an s ain, Ss160.03247+270. Animals om he h ee g oups
we e in amuscula ly injec ed (IM), and he i al dose used in he
g oups B and C was 2×105 TCID50/ ish. Th ee ish pe g oup we e
andomly collec ed a 24, 48, and 72h pos -inocula ion (p.i.),
and we e eu hanized by a MS-222 (Sigma-Ald ich) o e dose.
Indi idual samples o head kidney and pooled eye/b ain we e
asep ically eco e ed, and immedia ely ozen in liquid ni ogen,
and s o ed a −80°C un il used.
To minimize ish su e ing, ials we e accomplished in acco -
dance o he Spanish di ec i e (RD 1201/2005) o he p o ec ion
o animals used in scien i ic expe imen s, and by he Bioe hics
and Animal Wel a e Commi ee o he IFAPA o he egula-
ion o animal ca e and expe imen a ion (app o ed numbe
10-06-2016-102).
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Labella e al. T ansc ip omic Analysis o NNV-In ec ed Sole
F on ie s in Immunology | www. on ie sin.o g July 2018 | Volume 9 | A icle 1626
na ex ac ion and cDna syn hesis
O gans collec ed (50–100 mg) om inocula ed ish we e homog-
enized in 1mL o TRI Reagen ® (Sigma-Ald ich) using a mixe
mill MM400 (Re sch, Haan, Ge many) wi h a p og am o 20 /s
5min. A e 5min a oom empe a u e, a olume o 100µL o
1-b omo-3-chlo op opane (Applichem, Da ms ad , Ge many)
was added o he samples and o exed o 15s. A e 5min a
oom empe a u e, samples we e cen i uged a 12,000×g o
15min a 4°C, ob aining h ee phases. The uppe aqueous phase
was eco e ed and mixed wi h e hanol 75% (1:1). To con inue he
ex ac ion o o al RNA, RNeasy Mini ki (Qiagen GmbH, Hilden,
Ge many) was used acco ding o he manu ac u e ’s ins uc ions.
RNA samples we e esuspended in nuclease- ee wa e (Qiagen),
quan i ied by spec opho ome y a 260nm using a NanoD op
ND 1000 Spec opho ome e (NanoD op Technologies, Wes
Palm Beach, FL, USA), and s o ed a −80°C un il used. RNA sam-
ples we e ea ed wi h RNase- ee DNase I (Roche Diagnos ics
GmbH, Mannhein, Ge many), o a oid genomic DNA con ami-
na ion, ollowing manu ac u e ’s ins uc ions. Fo he syn hesis
o cDNA, T ansc ip o Fi s S and cDNA Syn hesis Ki (Roche)
was used ollowing manu ac u e ’s ins uc ions. cDNA samples
we e quan i ied a 260nm wi h he nanod op sys em, and s o ed
a −20°C un il used.
sample P epa a ion o na-seq and aw
eads Da a P ocessing
RNA-Seq was pe o med using he Illumina HiSeq™ 2000 pla -
o m (Cen o de Análisis Genómico, CNAG-CRG, Ba celona,
Spain). To selec he ime-poin o RNA-Seq analysis, an-
sc ip ion o Mx gene and i al eplica ion we e quan i ied using
p e iously desc ibed ampli ica ion p o ocols (25, 26). RNA
quali y/quan i y was checked ollowing he c i e ia p oposed
by CNAG-CRG, including a RNA in eg i y numbe RIN>8
(Agilen 2100 Bioanalyze , Agilen , Waldb onn, Ge many),
and abso bance a ios 260/280 and 260/230 o 1.8–2.0 (Figu e
S1 and Table S1 in Supplemen a y Ma e ial). RNA-Seq was
pe o med o ob ain>40 million pai ed-ended (PE) eads o
75bp leng h pe sample. RNA-Seq aw eads (FASTQ) (27) we e
p ocessed using bioin o ma ics ools o he Supe compu ing
and Bioinno a ion (SCBI) cen e o he Uni e si y o Malaga.
Da a we e empo ally s o ed a he Picasso SGI O igin 2000
supe compu e (SCBI).
P e-p ocessing o aw eads was pe o med using he
SeqT imNex ( 2.0.60) pipeline (SCBI) in o de o emo e low
quali y sequences, con aminan s, adap o s, ec o s, and o he
a i ac s p io o he assembly (28). The sc ip bow ie2 ( 2.2.9)
(29) was used o mapping and assembling he p e-p ocessed
FASTQ eads using as e e ence he Senegalese sole ansc ip-
ome da abase SoleaDB, .4.1 2014-01-231 (30). Finally, he sc ip
sam2coun s ( 20131126) (31) was used o con e SAM mapping
esul s in o e e ence sequence coun s.
These RNA-Seq da a ha e been deposi ed in he NCBI Gene
Exp ession Omnibus da abase wi h he expe imen al se ies
accession numbe : GSE101877.
1 www.scbi.uma.es/soleadb (Accessed: Ap il, 2017).
s a is ical analysis, iden i ica ion, and
anno a ion o Degs
To iden i y DEGs, cleaned, mapped, and coun ed da a se s
ob ained om expe imen al g oups B and C (animals inocula ed
wi h wSs160.03 and Ss160.03247+270 easso an s, espec i ely)
we e compa ed o he con ol g oup A (animals injec ed wi h
L-15 medium). The Bioconduc o package (including edgeR,
DESeq2, and limma app oaches), which uses R s a is ical
p og amming language,2 was applied o he analysis and com-
p ehension o high- h oughpu ansc ip omic da a (32–35). The
alse disco e y a e (FDR) was used o de e mine he h eshold
p- alue o mul iple es s (34). T ansc ip s wi h FDR alues lowe
han 0.05 (signi icance le el) we e conside ed as DEGs. Venn-
Diag am me hod was used o compa e and isualize da ase s
o expe imen al g oups wi h he h ee di e en Bioconduc o
app oaches (36). Fo he unc ional anno a ion o he ansc ip s,
Sma3s, Au oFac , and Full-Leng he Nex we e used o p o ide
gene desc ip ion (37–39). These anno a ion ools we e consul ed
om SoleaDB ansc ip ome da abase (30). In addi ion, Gene
On ology (GO) en ichmen analysis was ca ied ou using GO
da abase3 and genes wi h old change (FC) alues highe han 1.5
(ei he up- o down egula ed).
analysis o gene exp ession by
Quan i a i e eal-Time Pc (q T-Pc )
All he eac ions we e conduc ed using he Ligh Cycle 96
Te mocycle (Roche) and he Fas S a Essen ial DNA G een
Mas e Mix (Roche) using SYBR G een echnology. PCRs we e
ca ied ou in 20-µL mix u es con aining cDNA gene a ed
om 50ng o RNA, 10µL o Fas S a Essen ial DNA G een
Mas e 2×, 1µL o each p ime (10pmol) and 7µL o wa e .
Te mocycling condi ions we e: ini ial dena u a ion a 95°C o
10 min, ollowed by 45 ampli ica ion cycles o 95°C o 10 s,
60°C o 10s, and 72°C o 10s. To ob ain mel ing cu es, he
ollowing p o ile was conduc ed: 95°C o 10 s, 65°C o 60s,
and 97°C o 1s. Each sample was un in iplica e o analysis.
Eigh di e en ially exp essed genes we e andomly selec ed o
qRT-PCR e i ica ion. Ribosomal p o ein subuni 4 gene ( ps4)
was s ably exp essed h oughou he expe imen s and was used
as he in e nal con ol (Table S2 in Supplemen a y Ma e ial). The
ela i e exp ession a io o he a ge genes e sus ps4 gene was
calcula ed using 2−ΔΔCT me hod, and all da a we e gi en in e ms
o ela i e mRNA exp ession (40).
esUlTs
T ansc ip omic sequencing and aw
eads Da a P ocessing
T ansc ip ion o Mx gene and i al eplica ion we e quan i ied in
bo h head kidney and eye/b ain samples om each expe imen al
condi ion (L-15, wSs160.03, and Ss160.03247+270) a 24, 48, and
72h p.i. T ansc ip ion o Mx and i al eplica ion eached he
2 h ps://www.bioconduc o .o g/ (Accessed: Ap il, 2017).
3 www.geneon ology.o g (Accessed: Ap il, 2017).
Table 1 | Illumina sequencing analysis o issue Senegalese sole samples
in ec ed wi h ne ous nec osis i us easso an s, and L-15-con ol g oup, a
48h p.i.
sample To al aw
eadsa
To al clean
eadsb
% con aminan
sequences
emo ed
To al
unigenesc
head kidney
L-15 331,555,320 321,395,622 3.1 –
wSs160.03 269,346,896 263,149,296 2.3 28,954
Ss160.03247+270 303,553,076 296,703,761 2.3 28,964
eye/b ain
L-15 289,923,568 231,003,910 20.3 –
wSs160.03 310,151,374 245,611,206 20.8 27,135
Ss160.03247+270 293,997,192 238,197,453 19 27,491
aTo al numbe o ead-pai s (o eads o single-end uns) ha passed Illumina il e .
bTo al numbe o clean eads a e he analysis wi h he SeqT imNex pipeline.
cTo al numbe o unigenes a e mapping wi h bow ie2 using Solea DB ( .4.1 2014-
01-23) as e e ence ansc ip ome.
Table 2 | DEGs eco ded wi h edgeR om Solea senegalensis subjec ed o i al
in ec ion wi h ne ous nec osis i us easso an s a 48h pos in ec ion.
Up egula ed
genes (%)
Down egula ed
genes (%)
To al
Degs
Fca a ios (up-/
down egula ed)
wss160.03
Head kidney 358 (88) 49 (12) 407 14.2–12.3
Eye/b ain 206 (91) 20 (9) 226 5.2–3.2
To al 564 (89) 69 (11) 633
ss160.03247+270
Head kidney 129 (93) 10 (7) 139 11.6–12.4
Eye/b ain 28 (11) 226 (89) 254 5.3–3.5
To al 157 (40) 236 (60) 393
aRange o old-change alues o each easso an and o gan sampled.
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F on ie s in Immunology | www. on ie sin.o g July 2018 | Volume 9 | A icle 1626
highes s alues a 48h p.i., and nei he Mx ansc ip ion no i al
eplica ion was obse ed a 24h p.i. (da a non-shown). The e o e,
samples om 48h p.i. we e selec ed o RNA-Seq analysis ( h ee-
old eplica ed), being sequenced using Illumina HiSeq™ 2000
pla o m. Be ween 269,346,896 and 331,555,320 aw eads we e
ob ained om head kidney samples. These eads we e p e-p o-
cessed wi h he SeqT imNex pipeline, and be ween 2.3 and 3.1%
o con aminan sequences we e emo ed (Table1). In eye/b ain
samples, he ange o aw eads ob ained was be ween 289,923,568
and 310,151,374. A e he SeqT imNex p e-p ocessing, 20% o
con aminan sequences we e emo ed (Table1). A e he map-
ping and assembling o he p e-p ocessed eads wi h bow ie2
sc ip , using as e e ence he SoleaDB ansc ip ome ( .4.1 2014-
01-23), a o al numbe o 56,089 unigenes (28,954 om head
kidney and 27,135 om eye/b ain samples) we e ob ained o
he g oup in ec ed wi h he w , high i ulen isola e (wSs160.03).
Rega ding he g oup in ec ed wi h he mu an , less i ulen s ain
( Ss160.03247+270), 56,455 o al unigenes (28,964 om head kidney
and 27,491 om eye/b ain samples) we e ob ained (Table 1).
These esul s ep esen a di e ence on ansc ip de ec ion o
0.65% be ween animals inocula ed wi h bo h i uses.
iden i ica ion o Di e en ially
exp essed genes
To iden i y DEGs, ansc ip s de ec ed om animals inocula ed
wi h he w o he mu an s ains we e compa ed wi h hose
ob ained om he con ol g oup (L-15), using h ee s a is ical
app oaches o he Bioconduc o package (edgeR, DESeq2, and
limma). The analysis wi h edgeR was he mos e icien me hod,
de ec ing 1,026 DEGs (99.5% o 1,031 o al DEGs) (alone o
oge he wi h DESeq2) (Table2), whe eas wi h he applica ion o
DESeq2, only 359 DEGs (34.8%) (alone o oge he wi h edgeR)
we e ob ained (da a no shown). No DEGs we e ob ained wi h
he limma ool.
Based on he esul s ob ained wi h edgeR, a o al o 633 DEGs
we e de ec ed in animals inocula ed wi h he w easso an
(highly i ulen ), whe eas 393 DEGs we e de ec ed in animals
in ec ed wi h he mu an s ain, which means a educ ion o
37.9%. Rega ding up- and down egula ed DEGs, he pe cen -
ages be ween up- and down egula ed genes we e 89 and 11%,
espec i ely o he w isola e, while o he mu an s ain, he
pe cen ages we e 40 and 60%, espec i ely (Table2). Acco ding
o he o gans es ed, he pe cen ages o up- and down egula ed
DEGs in head kidney was simila o bo h i al s ains, ob aining
88 and 12% o wSs160.03, and 93 and 7% o Ss160.03247+270
(Table 2). Fo he eye/b ain samples, a simila p opo ion
be ween up-/down egula ed DEGs was ob ained a e in ec ion
wi h he w isola e (91 and 9%); howe e , an in e sion in he up-/
down egula ed DEGs p opo ion (11 and 89%) was ob ained
in eye/b ain samples om animals inocula ed wi h he mu an
s ain (Table2).
compa a i e analysis o Degs De ec ed
a e in ec ion Wi h bo h Vi al
easso an s
A compa a i e analysis has been pe o med in o de o de e mine
sha ed and di e en DEGs de ec ed be ween he in ec ion wi h
he w and he mu an s ains. F om he o al numbe o DEGs
de ec ed a e in ec ion wi h bo h i al s ains (1,026), 610 DEGs
(60.8%) we e exclusi ely de ec ed a e he in ec ion wi h he w
isola e, whe eas 370 DEGs (36.9%) we e exclusi ely de ec ed a e
in ec ion wi h he mu an s ain. The numbe o sha ed DEGs
ob ained be ween bo h i al s ains was only 23 (2.3%) (Figu e1),
mainly ela ed o unc ions such as, signal ansduc ion pa hways,
in lamma o y esponse, cellula in eg i y, and me abolism (Table
S3 in Supplemen a y Ma e ial).
Compa ing DEGs de ec ed om head kidney samples, 396
and 128 DEGs we e exclusi ely iden i ied a e he in ec ion wi h
he w and mu an s ains, espec i ely; whe eas, he numbe o
sha ed DEGs de ec ed was only 11, being 6 up egula ed and 5
down egula ed (Table S3 in Supplemen a y Ma e ial). In he case
o eye/b ain samples, a simila numbe o DEGs we e iden i ied
a e he in ec ion wi h w and mu an s ains (214 and 242,
espec i ely). The numbe o sha ed unigenes de egula ed was
only 12, being 2 up egula ed and 10 down egula ed (Figu e1).
Func ional anno a ion
In o de o s udy he biological unc ions o he iden i ied DEGs,
a GO en ichmen analysis was pe o med. Only unigenes wi h FC
Table 3 | Anno a ed unigenes in gene on ology da abase.
To al numbe
o unigenesa
numbe o anno a ed
unigenes (%)
numbe o di e en
on ologies de ec ed
wss160.03
Head kidney 319 269 (84.33) 411
Eye/b ain 211 113 (53.55) 282
To al 530 382 (72.07) 693
ss160.03247+270
Head kidney 135 73 (54.07) 202
Eye/b ain 161 110 (68.32) 566
To al 296 183 (61.82) 768
aTo al numbe o unigenes wi h old change highe han 1.5 (up- o down egula ed).
FigU e 1 | Venn diag am o DEGs using edgeR. Common and di e en
DEGs de ec ed a e in ec ion wi h he wild- ype and mu an easso an s
in head kidney and ne ous issue. Numbe o DEGs: up egula ed+
down egula ed.
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highe han 1.5 (up- o down egula ed) and p- alue<0.05 we e
conside ed. Fo he w easso an in ec ion samples, a o al o
530 unigenes we e de ec ed (319 in head kidney and 211 in eye/
b ain samples). In con as , in samples in ec ed wi h he mu an
s ain, lowe numbe s o unigenes (296) we e ob ained (135
and 161 in head kidney and in eye/b ain samples, espec i ely)
(Table 3). Di e en pe cen ages o GO da abase anno a ion o
he unigenes we e ob ained o he samples analyzed, wi h a
educ ion o 10.25% o anno a ed unigenes in he mu an s ain
in ec ion samples (Table3). Howe e , he numbe o associa ed
on ologies o each unigene was highe in he mu an in ec ed
samples. DEGs we e classi ied in o h ee subclasses: biological
p ocess (BP), cellula componen (CC), and molecula unc ion
(MF), and he 20 on ologies mo e equen ly de ec ed wi hin each
subclass a e ep esen ed in Figu es2 and 3, o he w and mu an
s ains, espec i ely.
Di e en on ology p o iles we e ob ained o each ype o i al
in ec ion. This esul was specially e idenced o he BP subclass;
speci ically, in head kidney samples, only “p o eolysis” and “ATP
ca abolic p ocess” we e sha ed in bo h i al in ec ion samples,
while no simila i ies we e obse ed in samples om eye/b ain. In
he case o CC and MF subclasses, simila p o iles we e de ec ed
be ween bo h i al in ec ion samples. The mo e equen on olo-
gies wi hin CC and MF subclasses in bo h o gans sampled, had a
clea ela ion o cellula memb ane and ex acellula ma ix wi h
an ene ge ic cos associa ed (“ATP binding”), and o calcium and
zinc-dependen enzyma ic ac i i ies (Figu es2 and 3).
Rega ding each i al in ec ion, in head kidney samples inocu-
la ed wi h he w easso an isola e, he mos equen ly de ec ed
on ologies we e “p o eolysis” (BP), “in eg al componen o mem-
b ane” (CC), and “ATP binding” (MF). In eye/b ain samples, he
p o ile ob ained was mo e homogeneous, a ying only “s uc u al
molecule ac i i y” in he MF subclass (Figu e2). Fo he samples
inocula ed wi h he mu an s ain, he mos p edominan on olo-
gies we e “p o eolysis” (BP), “in e media e ilamen ” (CC), and
“s uc u al molecule ac i i y” (MF) in head kidney samples; and
“ asculogenesis” (BP), “in eg al componen o memb ane” (CC),
and “calcium ion binding” (MF) in eye/b ain samples (Figu e3).
The unigenes associa ed wi h he mos equen ly de ec ed
on ologies wi hin he BP subclass, “p o eolysis,” and “ asculogen-
esis” a e de ailed in Table S4 in Supplemen a y Ma e ial. Rega ding
“p o eolysis,” 18 unigenes (9 in each o gan) we e de egula ed in
animals in ec ed wi h he w isola e (FC alues om −1.67 o
9.04 in head kidney and om 1.62 o 4.30 in eye/b ain samples),
whe eas 15 unigenes (12 in head kidney and 3 in eye/b ain sam-
ples) we e de egula ed in he case o he mu an s ain (FC alues
om 2.46 o 9.11 in head kidney and om −2.40 o 2.49 in eye/
b ain samples) (Table S4 in Supplemen a y Ma e ial). Cys eine
p o eases, se ine p o eases, and me allop o eases we e he main
enzyme amilies de ec ed. In con as , “ asculogenesis” was only
de ec ed in eye/b ain samples o animals in ec ed wi h he mu an
s ain. Fo his on ology, eigh unigenes we e de ec ed, being all
down egula ed wi h FC alues anging om −2.69 o −1.53
(Table S4 in Supplemen a y Ma e ial).
immune esponse o senegalese sole
agains Vi al easso an in ec ions
Sha ed DEGs in samples o bo h o gans es ed h we e iden i ied
a e in ec ion wi h bo h i al s ains. Fo he w isola e, 42 DEGs
(6.6% ou o 633) we e de ec ed in bo h o gans. Mos o hese
DEGs we e up egula ed, excep o COL1A2, which is in ol ed
in in lamma o y esponse and signaling pa hways and, which
was down egula ed in bo h o gans. Rega ding up egula ed
DEGs, a clea ela ionship wi h he inna e immune esponse
agains i al in ec ion was obse ed, being unigenes de ec ed
hose coding pa e n ecogni ion ecep o s (PRRs) (DHX58), and
IFN-s imula ed genes (ISG15, Mx, STAT1, HERC5, IFI44, IFIT-1,
NUP133, and TRIM21). In addi ion, unigenes ela ed o he ol-
lowing cellula unc ions we e also up egula ed: (i) apop osis and
cell p oli e a ion (MACPF, u-PAR, PARP14, EPSTI1, and ISG12);
(ii) an igen p ocessing and p esen a ion (RNF213, HERC4, and
MHC class II genes); (iii) signaling pa hways (ANXA3 and RTP3);
(i ) in lamma o y esponse (CCL19L1); and ( ) cy oskele on
and ex acellula ma ix (SMCHD1 and ACTB) (Table S5 in
Supplemen a y Ma e ial). In e es ingly, only 3 DEGs (0.8% ou o
393) (COL1A2, RDH13, and RPS12), we e de ec ed in samples o
bo h o gans a e inocula ion wi h he mu an s ain, all o hem
down egula ed (Table S5 in Supplemen a y Ma e ial) and ela ed
mainly o signaling pa hways and in lamma o y esponse.

FigU e 2 | Gene on ology en ichmen analysis o di e en ially exp essed genes om head kidney (a) and eye/b ain (b) samples o Senegalese sole animals
in ec ed wi h he wild- ype easso an . On ologies we e classi ied in o h ee subclasses, including biological p ocess, cellula componen , and molecula unc ion.
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FigU e 3 | Gene on ology en ichmen analysis o di e en ially exp essed genes om head kidney (a) and eye/b ain (b) samples o Senegalese sole animals
in ec ed wi h he mu an easso an . On ologies we e classi ied in o h ee subclasses, including biological p ocess, cellula componen , and molecula unc ion.
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The analyses o genes exp essed a e in ec ion wi h each o
i al isola e also allow us o de ec up egula ion o ype I IFN
(IFN I) genes exclusi ely in animals in ec ed wi h he w isola e,
including genes encoding o PRRs, media o s o IFN signaling
cascades, and IFN inducible p o eins (Table S6 in Supplemen a y
Ma e ial). In addi ion, o he genes ela ed o he an i i al esponse
ha e also been up egula ed (FC>10) in head kidney samples
o Senegalese soles inocula ed wi h he w isola e, such as genes
ela ed o p o ein ubiqui ina ion (MAGEL2, FC: 11.08); an igen
p ocessing and p esen a ion (GILT, FC: 11.08); i us esponsi e
genes (VGR) (He pes gp2 mul i-domain p o ein, FC: 11.08;
claudin-like p o ein ZF-A89, FC: 10.21); in lamma o y esponse
genes (4F2 cell-su ace an igen hea y chain-like, FC: 9.9); immune
e ec o s (ca hepsin Z, FC: 9.04); and genes ela ed o apop osis
(BIRC5, FC: 11.22; ho GTPase-ac i a ing p o ein 11A, FC: 7.75;
and -e s e y h oblas osis i us E26 oncogene homolog 1, FC: 8.27)
(Table S6 in Supplemen a y Ma e ial). Fu he mo e, se e al
unigenes ela ed o C- ype lec ins we e mainly iden i ied in he
ne ous issue (Table S6 in Supplemen a y Ma e ial).
In con as , he mu an s ain did no induce DEGs ela ed o
IFN I nei he in head kidney no in ne ous issue; howe e , DEGs
ela ed o C- ype lec ins ecogni ion and signaling mechanisms
we e up egula ed in head kidney samples. In addi ion, se e al
unigenes wi h a ole in he immune esponse o Senegalese soles
we e de ec ed, especially hose in ol ed in di e en signaling
pa hways (JNK, TLRs, p o ein ubiqui ina ion, G-p o ein), and
in lamma o y esponse (Table S7 in Supplemen a y Ma e ial).
These DEGs we e down egula ed in eye/b ain samples, unlike i
was obse ed a e in ec ion wi h he w isola e, whe e mos o he
DEGs we e up egula ed.
Ve i ica ion o Degs by q T-Pc
To u he con i m he ansc ip omic sequencing, he exp ession
p o iles o eigh andomly selec ed DEGs (including genes ela ed
o di e en pa hways, up o down egula ed, de ec ed a e in ec-
ion wi h bo h i al s ain, and bo h o gan sampled) we e meas-
u ed by qRT-PCR. As shown in Figu e4, he qRT-PCR esul s
e ealed simila exp ession endency as he high- h oughpu
sequencing da a, despi e some quan i a i e di e ences a he
exp ession le el, con i ming ha exp ession o DEGs de ec ed
by RNA-Seq is happening. In addi ion, unigenes up and down-
egula ed we e adjus ed wi h an R2=0.91 demons a ing a good
co ela ion be ween bo h echniques.
DiscUssiOn
Immune esponse agains be anoda i us in ec ions has been
poo ly s udied. In o de o imp o e he knowledge o be ano-
da i us pa hogenesis in Senegalese sole, wo i al s ains wi h
di e en deg ee o i ulence ha e been es ed, wSs160.03 a na u-
al RGNNV/SJNNV easso an highly i ulen o Senegalese
sole (14), and a lowe i ulen isola e, he mu an easso an
( Ss160.03247+270) ha bo ing mu a ions a posi ions 247 and 270
in he capsid p o ein. These mu a ions esul ed in decay o 40%
in he ish mo ali y a e, indica ing ha hese amino acidic
posi ions would ha e an impo an e ec on i ulence (14, 41).
In his s udy, we ha e applied he RNA-Seq echnology o s udy
he possible ole o hese i ulence de e minan s in he immune
esponse o Senegalese sole ju eniles.
This is he i s s udy ha applies a RNA-Seq massi e sequenc-
ing s a egy o ob ain he whole ansc ip ome o Senegalese sole
a e i al in ec ions. In ac , as a as we know, un il now, he e
a e jus wo s udies applying RNA-Seq o de e mine Senegalese
sole ansc ip ome (30, 42). Howe e , none o hese au ho s
s udied he implica ion o i al in ec ions on Senegalese sole
ansc ip ome.
The me hodology used o p e-p ocessing and assembling he
Illumina aw eads allowed us o de ec almos he same numbe
o unigenes (di e ences lowe han 1%) o each i al easso an
and issue analyzed, which p o ides a high deg ee o consis ency
in he subsequen compa a i e analyses pe o med. A sligh ly
lowe numbe o unigenes was de ec ed in ne ous issue (eye and
b ain), p obably because o he high pe cen age o con amina ed
sequences emo ed om his issue (Table1) ha could be due
o he own na u e o he issue.
A highe numbe o DEGs was de ec ed a e in ec ion wi h
he high i ulen easso an isola e (Table 1). Pu cell e  al.
(20), using isola es wi h high- and lowe - i ulence o in ec ious
haema opoie ic nec osis i us, epo ed ha he mos i ulen
isola e induced a highe hos ansc ip omic change in ainbow
ou (20). Fu he mo e, he p opo ion o sha ed genes induced
by bo h i al easso an s ains was qui e low (2.3%) (Figu e1),
compa ed wi h he numbe o genes exp essed di e en ially by
bo h easso an s. I is also ema kable ha a e he inocula ion
wi h he mu an s ain, he numbe o DEGs was lowe in head
kidney samples, being he numbe o down egula ed genes highe
in ne ous issues (Table 2). The in e sion in he p opo ion o
up/down egula ed DEGs obse ed in ne ous issue, he NNV
a ge o gan, a e in ec ion wi h he mu an s ain could suppo
he hypo hesis ha mu a ions a aminoacid 247 and 270 p o oke
con o ma ional changes in he i al capsid ha could esul in a
loss o a ini y o he hos cell ecep o s o in a di e en opism
o he i us (41).
The on ology analyses indica e a iabili y be ween he ypes
o i al s ains and issues. Thus, he di e en numbe o on olo-
gies de ec ed o bo h easso an s akes special ele ance i we
compa e hem wi h he numbe o o al unigenes de ec ed o
each i al s ain. This esul could e lec a mo e speci ic hos
esponse o in ec ion wi h he w isola e (693 on ologies asso-
cia ed wi h 530 unigenes) in con as o ha obse ed o he
mu an s ain (768 on ologies associa ed wi h 296 unigenes)
(Table 3). The main on ologies de ec ed in o he subclasses o
CC and molecula unc ion o bo h i al s ains we e “in eg al
componen o memb ane,” “ATP binding,” “zinc ion binding,” “Ca
ion binding,” o name he mos equen ly eco ded. Al hough
he e is an ini ial ene gy-dependen ecogni ion o he i al
pa hogen h ough he ish cellula memb ane by means o Ca2+
and Zn2+, he signal ansduc ion mechanisms igge ed a e
his ini ial p ocess a e comple ely di e en in bo h easso an s,
de i ing in he ac i a ion o di e en biological p ocesses in he
hos cell. The con o ma ional changes p o oked by he mu a ions
a amino acids 247 and 270 o he i al capsid may esul in he
induc ion o di e en ansduc ional pa hways, leading o di e -
en biological p ocess: immune esponse and p o eolysis o he
FigU e 4 | Valida ion o RNA-Seq da a by quan i a i e eal- ime PCR (qRT-PCR). (a) Compa ison o he exp essions p o ile o eigh DEGs de e mined by Illumina
HiSeq™ 2000 sequencing pla o m and qRT-PCR a 48h (pi) using ibosomal p o ein subuni S4 ( ps4) as housekeeping gene. Da a shown a e he mean o
iplica es±SD. (b) Co ela ion o da a be ween RNA-Seq and qRT-PCR echniques.
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w isola e, and p o eolysis and asculogenesis inhibi ion o he
mu an easso an .
I is known ha NNV causes apop osis in i s hos (43). In ou
s udy, cell apop osis, iden i ied as p o eoly ic clea age o cellula
p o eins, was de ec ed in animals in ec ed wi h he w isola e, as
well as in head kidneys o animals in ec ed wi h he mu an s ain.
The p o eoly ic clea age includes he pa icipa ion o ca hepsins,
se ine p o eases, calpains, and me allop o eases. The in ec ion
wi h he mu an s ain p o okes down egula ion o genes ela ed
o asculogenesis in ne ous issue, p o oking inhibi ion o as-
culogenesis, and u he apop osis (44, 45). These esul s indica e
ha bo h i al s ains induced apop osis, al hough h ough di -
e en mechanisms.
Di e en ially exp essed genes and on ology analyses e idence
ha immune esponse is a biological p ocess mainly egula ed in
head kidney o ish inocula ed wi h he w isola e, highligh ing
he inna e immune esponse media ed by IFN I. Vi al nucleic
acid is de ec ed in he hos cells by PRRs, being RLR and MyD88-
dependen TLR signaling pa hways ound o media e ype I
IFN induc ion in esponse o RNA i us in ec ion (46). The
RLR pa hway is composed o h ee RNA cy oplasmic senso s,
such as RIG-I, MDA5, and LGP2. In he p esen s udy, only