Immune responses of orange-spotted grouper, Epinephelus

Research paper Yu-Xiong Laia, Bao-Lei Jina, Yu Xua, Li-jie Huanga, Run-Qing Huanga, Yong Zhanga, Jimmy Kwangb,∗∗, Jian-Guo Hea,c, Jun-Feng Xiea,∗ a State Key Laboratory of Biocontrol/MOE

Research paper

Yu-Xiong Laia, Bao-Lei Jina, Yu Xua, Li-jie Huanga, Run-Qing Huanga,

Yong Zhanga, Jimmy Kwangb,∗∗, Jian-Guo Hea,c, Jun-Feng Xiea,∗

a State Key Laboratory of Biocontrol/MOE Key Laboratory of Aquatic Product Safety, School of Life Sciences, Sun Yat-sen University,

Guangzhou 510275, China

b Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, 117604 Singapore, Singapore

c School of Marine Sciences, Sun Yat-sen University, Guangzhou 510275, China

a r t i c l e i n f o

Article history:

Received 28 May 2013

Received in revised form 7 October 2013

Accepted 8 October 2013

Keywords:

Betanodavirus

Virus-like particle

Humoral immune response

Cellular and innate immune responses

Vaccine

Grouper

a b s t r a c t Betanodavirusesarethecausativeagentsofviralnervousnecrosis(VNN),aseriousdisease

ofculturedmarinefishworldwide.Virus-likeparticles(VLPs)areoneofthegoodnovel vaccinecandidatestocontrolthisdisease.Untilnow,betanodavirusvaccinestudiesmainly focusedonthehumoralimmuneresponseandmortalityafterviruschallenge.However, littleisknownabouttheactivationofgenesresponsibleforcellularandinnate immu-nitybyvaccines.Inthepresentstudy,VLPsoforange-spottedgroupernervousnecrosis virus(OGNNV)wereproducedinprokaryotesandtheirabilitytoenterAsianseabasscells wasthesameasnativevirus,suggestingthattheypossessasimilarstructuretoOGNNV VLPsimmunogenicitywasthendeterminedbyintramuscularlyvaccinatingEpinephelus coioidesatdifferentconcentrations(1.5or15␮gg−1fishbodyweight,FBW)and immu-nizingfrequencies(administrationonce,twiceandthrice).Asinglevaccinationwiththe dosageof1.5␮gg−1FBWisenoughtoprovokehightiterantibodies(average3foldhigher thanthatofnegativecontrol)withstrongneutralizingantibodytiterasearlyas1weekpost immunization.Furthermore,quantitativePCRanalysisrevealedthatelevengenes associ-atedwithhumoral,cellularandinnateimmunitieswereup-regulatedintheliver,spleen andheadkidneyat12hpostimmunization,correlatingwiththeearlyantibodyresponse

Inconclusion,wedemonstratedthatVLPvaccinationinducedhumoralimmuneresponses andactivatedgenesassociatedwithcellularandinnateimmunityagainstbetanodavirus infectioninorange-spottedgrouper

© 2013 Elsevier B.V All rights reserved

1 Introduction

∗ Corresponding author Tel.: +86 20 39332970; fax: +86 20 84113229.

∗∗ Corresponding author Tel.: +65 68727470; fax: +65 68727007.

E-mail addresses: kwang@tll.org.sg (J Kwang), xiejf@mail.sysu.edu.cn

(J.-F Xie).

0165-2427/$ – see front matter © 2013 Elsevier B.V All rights reserved.

from orange-spotted grouper, Epinephelus coioides (Ep.

Tanakaet al., 2008; Yuasaet al., 2002) It was

Yamashitaetal.,2009a,b)andAsianseabass(Pakingking

betan-odaviruses

Charlineetal.,2011).Theoretically,theyarenotinfectious

cheaper

investi-gated

2 Materials and methods

-GGAATTCAT-

(Fenneretal.,2006a).Cellswerewashedwith

(Fenneretal.,2006b)todeterminetheantibodytitersof

the2−CTmethod.Theexpressiondataofelevengenes

3 Results

Table 1

List of primers used in this work.

Primers used in vector construction

Primers used in real-time quantitative PCR

monomersinsuitablesituation,weresuccessfullypurified

bysucrose gradient ultracentrifugation aftersonication

Thereweretwo bandsata densityof1.07gcm−3

(frac-tionI)and1.13gcm−3(fractionII)intheultracentrifuge

tube,asshowninFig.1A.ThesizeofVLPsinfractionIIwas

Fig 1. Characterization of gradient-purified OGNNV VLPs (A) Two frac-tions of VLPs (F I and F II) were observed in sucrose gradient purification Electron microscopy of negatively stained VLPs (including fraction I and II) and OGNNV (WT) are shown (bar = 100 nm) (B) SDS-PAGE and Immunoblotting of VLPs and OGNNV As indicated, a strong protein band could be found at 37 kD and a weak band at 111 kD (maybe CP trimer)

in SDS-PAGE These two bands of proteins were verified as OGNNV CP by immunoblotting (IB) with mouse anti-VLP antibody (M = protein marker).

Fig 2.OGNNV and VLP invasion of SB cells observed by confocal microscopy SB cells were incubated with OGNNV or VLPs at 0◦C for 1 h and switched to

26◦C after a thorough wash At different time points post-incubation, OGNNV or VLPs were labeled with green fluorescence by IFA using self-made mouse anti-VLPs antibody as primary antibody and Alexa fluor 488-labeled goat anti-mouse IgG antibody while the cell nuclei were counterstained in blue with Hoechest White arrows indicate the attachment of particles to the cell membrane while red arrows show the gathering of particles near the cell nucleus The invasion track and timing of VLPs are the same as that of OGNNV (bar = 25 ␮m).

Tanakaetal.,2008),indicatingthatthe3Dstructureofthe

Fig 3. Humoral immune responses of orange-spotted grouper stimulated

by injecting IM VLPs VLPs in dosages of 15 ␮g g −1 FBW (high concentra-tion, V.H.) and 1.5 ␮g g−1FBW (low concentration, V.L.) were injected IM into Ep coioides (40 fish per group) once (A), twice (B) and thrice (C) with

a one-week-interval between multiple boosters PBS was used as neg-ative control Antisera from ten randomly selected fish were collected every week after the last immunization and subjected to antigen-capture ELISA The weeks post injection represent the time elapsed from the last immunization Absorbance values at each sampling time were the mean

of ELISA triplicate readings from 10 individual and antibody titers (fold change) represents that the data from vaccinated fish were normalized with that of PBS injected fish The error bars indicate standard deviation The asterisks indicated that the antibody responses of V.L and V.H were significantly higher than that of PBS control (**p < 0.01).

Fig 4.Neutralizing titers of anti-VLP sera from groupers Sera from

groupers immunized with one shot of V.L at 1, 7 and 10 weeks PI were

collected, serially three-fold diluted with MEM medium, premixed with

OGNNV (10 7 TCID 50 ml−1) in an equal volume and incubated with SB cells.

After culturing for 3 days, media were removed and IFA was performed to

determine the virus infection in cells The neutralizing titers (mean) were

shown above the bars and the error bars indicate standard deviation.

Pakingkinget al.,2009).Here, weinvestigated the

Table 2

Neutralization titer of anti-VLP sera from grouper.

Sera from groupers immunized with one shot of V.L at 1, 7 and 10 weeks PI were collected, serially three-fold diluted with MEM medium, premixed with OGNNV (10 7 TCID 50 ml−1) in an equal volume and incubated with SB cells After culturing for 3 days, media were removed and IFA was per-formed to determine the virus infection in cells The neutralizing potency (antibody titer) was calculated according to Reed and Muench (1938) and Spearman–Karber method ( Hamilton et al., 1977 ).

4 Discussion

2011;PlantandLaPatra,2011).Inthisstudy,OGNNVVLPs

Fig 5. Transcriptional changes of immune-related genes in five organs after VLPs immunization on Ep coioides VLPs (1.5 ␮g g−1FBW) were IM injected once into Ep coioides with PBS as control (60 fish per group) The expression raw data of 11 genes and 18S control were determined by SYBR green method of QPCR Data were analyzed using the 2 −CT method Fold units were calculated by dividing the expression values of vaccinated tissues by that of control once normalized with the 18S expression Values

at each indicated sampling time are the mean of triplicate readings The

(Pakingkinget al.,2010)andAsianseabass(Pakingking

2010;Groveetal.,2006)

2011;Utkeetal.,2007;Boudinotetal.,2001;Landisetal.,

2008;Parketal.,2009), indicatingthatthesemolecules

andCresswell,2001;Lenschowetal.,2007).During

etal.,2006;Loetal.,2004;Gaoetal.,2008).HSP90ABwas

etal.,2007;BurchandWeller,2005;Chaseetal.,2008),but

1992)

future

Competing interests

inter-ests

Author contribution

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