Protection of olive flounder, Paralichthys olivaceus, against viral hemorrhagic septicemia virus

Protection of olive flounder,Paralichthys olivaceus ,against viral hemorrhagicsepticemia virus (VHSV)by immunization with NV gene-knockout recombinant VHSVMin Sun Kim,Ki Hong Kim ⁎Department of Aquatic Life Medicine,Pukyong National University,Busan 608-737,South Koreaa b s t r a c ta r t i c l e i n f o Article history:Received 11January 2011Received in revised form 28January 2011Accepted 31January 2011Available online 5March 2011Keywords:Viral hemorrhagic septicemia virus Reverse genetics rVHSV-ΔNV-EGFP Olive flounder Vaccine potentialThe potential of the NV gene-knockout recombinant viral hemorrhagic septicemia virus (rVHSV-ΔNV-EGFP)as a live attenuated vaccine was assessed.In an in vivo virulence experiment,olive flounder (Paralichthys olivaceus )intramuscularly (i.m.)injected with 103and 105PFU of rVHSV-ΔNV-EGFP showed no mortality.The rVHSV-ΔNV-EGFP had ability to spread and replicate in the internal organs of olive flounder reared at either 15°C or 20°C.In the vaccination experiment,the cumulative mortality rates of olive flounder i.m.immunized with 103,104,and 105PFU of rVHSV-ΔNV-EGFP were 40–50%,10–30%,and 0–10%,respectively,at 4weeks after the primary immunization.In the second challenge experiment conducted at 4weeks after the boost immunization,the cumulative mortality in fish immunized with 103PFU of rVHSV-ΔNV-EGFP was 10–30%.No mortality was recorded in fish immunized with 104and 105PFU of the recombinant virus.While the cumulative mortality of fish in control group was 90–100%.The serum neutralization titers were also proportional to the survival rates of vaccine groups.The present results suggest that the rVHSV-ΔNV-EGFP has a high potential to be used as a live vaccine in olive flounder farms.©2011Elsevier B.V.All rights reserved.1.IntroductionViral hemorrhagic septicemia virus (VHSV),an enveloped nega-tive-strand RNA virus belonging to the genus Novirhabdovirus of the family Rhabdoviridae (Lenoir and de Kinkelin,1975;Walker et al.,2000;Tordo et al.,2005),causes a fatal disease resulting in mass mortality in a wide range of freshwater and marine fish species worldwide (Schlotfeldt and Ahne,1988;Schlotfeldt et al.,1991;Mortensen et al.,1999;Isshiki et al.,2003;Skall et al.,2005).Genetic analysis revealed four major genotypes of VHSV isolates (Snow et al.,1999;Einer-Jensen et al.,2004),and epidemics of each genotype have been correlated with the geographic location.Furthermore,it has been demonstrated that the virulence of VHSV depends on the virus genotype and the host species (Snow et al.,2005;Brudeseth et al.,2008).In Korea,only genotype IV of VHSV has been identi fied and has been one of the major culprits of high mortalities in farmed olive flounder (Paralichthys olivaceus )since firstly reported at 2001(Kim et al.,2003,2009).Thus development of affordable control measures against VHS is strongly needed to enhance productivity of olive flounder aquaculture.Vaccine-mediated prophylactic has been the most ef ficient and cost effective mean to control viral diseases.In fish rhabdoviral diseases,genetic vaccines based on the viral G gene expressingplasmids have been reported to have high protective ef ficacy against infectious hematopoietic necrosis virus (IHNV)and VHSV in rainbow trout and Atlantic salmon (Traxler et al.,1999;Lorenzen et al.,2001,2002;Lorenzen and LaPatra,2005;Chico et al.,2009;Einer-Jensen et al.,2009).Recently,live recombinant viral vaccines made by reverse genetics method,in which infectious negative-stranded RNA viruses are recovered entirely from cloned cDNA,have been attracted as a promising vaccine type (Schnell et al.,1994;Palese et al.,1996;Neumann et al.,2002;Neumann and Kawaoka,2004;von Messling and Cattaneo,2004).The vaccine potential of recombinant fish rhabdoviruses was firstly assessed by Thoulouze et al.(2004),in which rainbow trout intraperitoneally (i.p.)immunized with the recombinant IHNV carrying GFP gene instead of G gene were protected against wild IHNV ter,Romero et al.(2008)reported that i.p.immunization of rainbow trout with two recombi-nant IHNVs-rIHNV GFP (replaced NV gene with GFP gene)and rIHNV-Gvhsv GFP (replaced IHNV G gene with VHSV G gene),also,resulted in high protection against both IHNV and VHSV challenges.These results suggested that NV-de ficient IHNV could be used as an effective vaccine against rhabdoviral diseases in fish.To date,however,there are no reports on the vaccine potential of recombinant VHSVs.Lately,as all the previously rescued recombinant VHSVs (Biacchesi et al.,2010;Ammayappan et al.,2010)were originated from freshwater fish,we had generated a recombinant VHSV originated from olive flounder by replacement of the NV gene with the enhanced green fluorescent protein (EGFP)gene (rVHSV-ΔNV-EGFP).In the present study,we assessed the vaccine potential of the recombinant VHSV to be used as a live attenuated vaccine.The results showed thatAquaculture 314(2011)39–43⁎Corresponding author at:Department of Aquatic Life Medicine,Pukyong National University,Nam-gu 599-1,Busan 608-737,South Korea.Tel.:+82516295943;fax:+82516295938.E-mail address:khkim@pknu.ac.kr (K.H.Kim).0044-8486/$–see front matter ©2011Elsevier B.V.All rights reserved.doi:10.1016/j.aquaculture.2011.01.050Contents lists available at ScienceDirectAquaculturej o ur n a l h o m e p a g e :ww w.e l s ev i e r.c o m/l o c a t e /a q u a -o n l i n ethe NV deficient recombinant VHSV was non-pathogenic to olive flounder and conferred high protection against wild-type VHSV challenge by intramuscular immunization offish.2.Materials and methods2.1.Cells and virusEpithelioma papulosum cyprini(EPC)cells derived from carp (Cyprinus carpio L.)epidermal herpes-virus-induced hyperplasia lesions(Fijan et al.,1983)were grown in Leibovitz medium(L-15, Sigma)supplemented with penicillin(100U/ml),streptomycin (100μg/ml)and10%fetal bovine serum(FBS,Gibco).VHSV KJ2008 isolated in2008from moribund oliveflounder in a natural outbreak of VHS disease on a commercial farm in Korea(wild-type VHSV)was identified as genotype VI of VHSV by sequencing of the N gene and G gene.The wild-type VHSV and the recombinant virus,rVHSV-ΔNV-EGFP(submitted for publication;the procedures for rescue of recombinant VHSV are not shown here),were propagated in monolayer of EPC cells at15°C in the presence of2%FBS and antibiotics.Cultures displaying extensive cytopathic effect(CPE)were harvested and centrifuged4000×g for10min at4°C,and the supernatants were stored at−80°C.The titer of the wild-type VHSV and the recombinant VHSVs used in this study was determined by plaque assay(Burke and Mulcahy,1980).2.2.In vivo virulence of recombinant VHSVsTo compare virulence between the wild-type VHSV and the recombinant VHSV,oliveflounderfingerlings(4–5g;confirmed free-from VHSV and showing no signs of illness)were infected by intramuscular(i.m.)injection with the wild-type VHSV KJ2008or recombinant VHSV(rVHSV-ΔNV-EGFP)at two doses(103and105PFU/fish),and controlfish were injected with an equal volume of L-15alone. Experimentalfish in each group(10fish)were kept in six30L tanks at 15°C and mortalities were recorded daily for30d post-injection.2.3.In vivo replication of recombinant VHSVsTo estimate in vivo replication of the rVHSV-ΔNV-EGFP,olive flounderfingerlings reared in50L tanks at15°C or20°C were i.m. injected with the recombinant virus at a dose of105PFU/fish.At various time points(6h,12h,1d,2d,3d,5d,7d,and15d)after the injection,the spleen,kidney and liver were sampled from3fish in each group,and total RNA was extracted using RNAiso plus from each excised organ.The PCR primer pair sequences for the target G gene are:forward primer;5′-GTTTGTACAACATCACCCTGCCCA-3′,reverse primer;5′-TTGGAGACGAACACAATGCTCGGA-3′,and for a control18S ribosomal RNA gene are:forward primer;5′-CAAGACGGACGAAAGC-GAAAGCAT-3′,reverse primer;5′-TGGCATCGTTTACGGTCGGAACTA-3′.Thermal cycling conditions were1cycle of3min at95°C(initial denaturation)followed by18cycles(for18S rRNA)or35cycles(for VHSV G)of30s at95°C,30s at60°C,30s at72°C,with afinal extension step of7min at72°C.PCR samples to be compared were electrophoresed on the1%agarose gel,and stained with EtBr.2.4.Protection offish by immunization with rVHSV-ΔNV-EGFPTwo hundred forty oliveflounderfingerlings(4–5g)were randomly divided into4groups of two replicates,and reared in eight250L tanks(30fish/tank)at20–22°C.Fish were fed with a commercial pellet and acclimatized for2weeks before being immunized.Prior to immunization and blood sampling,thefish were starved for24h and anesthetized with MS222(Sigma)just before injection of the vaccine.Fish in each experimental group were immunized by i.m.injection with the rVHSV-ΔNV-EGFP at three doses (103,104,or105PFU/fish),andfish in the control group were injected with the L-15culture medium.At3weeks post-immunization,half of thefish in each group were transferred to tanks equipped with a refrigerating apparatus,and were adapted to15°C by gradual decrease of water temperature.At4weeks post-immunization,the fish adapted to15°C were challenged by i.m.injection of the wild-type VHSV KJ2008(103PFU/fish),and the remained half of thefish in each group were boost immunized with the same regime of the primary immunization.At3weeks post-boosting,fish were trans-ferred to refrigerating tanks and gradually adapted to15°C over a week.At4weeks post-boosting,6fish in each tank were bled to obtain serum,and the remnantfish were intramuscularly challenged with the pathogenic wild-type VHSV KJ2008at103PFU/fish.Mor-talities in thefirst and the second challenge experiments were recorded daily for30d post-challenge.2.5.Neutralization testFor neutralization test,the sera were previously heat inactivated at 56°C for30min to inactivate complement.The serially diluted sera (50μl/well)were mixed with an equal volume of wild-type VHSV (103PFU)in U-shaped96-well plates,and incubated at15°C for1h. Then,100μl of each mixture was added to triplicate wells containing EPC cells monolayer,and observed everyday CPE.The titer of each serum was the last dilution at which CPE was not observed.2.6.Statistical analysesSerum neutralization data were analyzed by the Student's t-test. Significant differences were determined at P b0.05.3.Results3.1.In vivo virulence of recombinant VHSVsThe cumulative mortality rates offish i.m.injected with the wild-type VHSV were90–100%,which was irrespective of the challenge doses(Fig.1).In contrast,nofish died by i.m.injection with rVHSV-ΔNV-EGFP at all challenged viral doses(Fig.1).3.2.Replication of rVHSV-ΔNV-EFGP in oliveflounder kept at15°C and20°CThe replication of rVHSV in i.m.infected oliveflounder was analyzed by semi-quantitative RT-PCR.The rVHSV-ΔNV-EGFP infish kept at15°C was detected at6h post-infection in all the examined organs(Fig.2a).The density of viral bands in the spleen and theliverFig.1.In vivo virulence of recombinant VHSV.Oliveflounder(Paralichthys olivaceus) were infected by intramuscular injection with the wild-type VHSV KJ2008(wtVHSV)or rVHSV-ΔNV-EGFP(rVHSV-EGFP)at two doses(103and105PFU/fish).Cumulative mortalities were recorded daily for30d post-injection.40M.S.Kim,K.H.Kim/Aquaculture314(2011)39–43was increased according to the lapse of time till 1–2d,then sharply decreased,and hardly detected at 15d post-infection.However,in the kidney,strong viral bands were shown till 3–5d post-infection.In fish maintained at 20°C,thick viral bands were seen in the spleen and the liver till 2–3d,and in the kidney till 1d (Fig.2b).3.3.VaccinationIn the first challenge experiment conducted at 4weeks after the primary immunization,the cumulative mortality rates in fish immu-nized with 103,104,and 105PFU of rVHSV-ΔNV-EGFP were 40–50%,10–30%,and 0–10%,respectively (Fig.3a).The cumulative mortality of fishin control group was 90–100%.In the second challenge experiment conducted at 4weeks after the boost immunization,the cumulative mortality in fish immunized with 103PFU of rVHSV-ΔNV-EGFP was 10–30%.However,no mortality was recorded in fish immunized with 104and 105PFU of the recombinant virus (Fig.3b).3.4.Serum neutralization activityThe serum neutralization titer of fish immunized with 105and 104PFU was signi ficantly higher than that of fish immunized with 103PFU (Fig.4).The serum of fish in the control group showed no neutralizationactivity.Fig.2.In vivo replication of rVHSV-ΔNV-EGFP.Olive flounder reared in either at 15°C (a)or at 20°C (b)were intramuscularly infected with 105PFU/fish of rVHSV-ΔNV-EGFP.At various time points (6h,12h,1d,2d,3d,5d,7d,and 15d)after the injection,the spleen,kidney and liver were sampled from 3fish in each group,and analyzed the ampli fication of transcripts corresponding to VHSV G gene and olive flounder's 18S ribosomal RNA gene by semi-quantitative RT-PCR.41M.S.Kim,K.H.Kim /Aquaculture 314(2011)39–434.DiscussionThe role of the NV gene in virulence of novirhabdoviruses is inconsistent according to viral species.Alonso et al.(2004)reported that zebra fish challenged with a recombinant SHRV lacking NV gene ORF did not show any statistical difference in cumulative mortalities compared to fish challenged with wild type SHRV.However,in IHNV,the recombinant virus lacking NV gene ORF did not induce any disease-related symptoms and any mortality in rainbow trout challenged through either immersion or injection (Thoulouze et al.,2004).Ammayappan et al.(2010)demonstrated that the virulence of VHSV in yellow perch was highly decreased by substitution of NV ORF with EGFP ORF.In this study,the final cumulative mortality in the group of control fish was 90–100%.However,olive flounder challenged by rVHSV-ΔNV-EGFP showed no mortality even at high doses as in the NV-de ficient rIHNV (Thoulouze et al.,2004).These results suggest that NV protein of VHSV is deeply related to virulence on olive flounder,and NV gene knockout-mediated attenuation of VHSV can be a potential way to produce a live viral vaccine against VHS disease in olive flounder.The level of in vivo replication and duration of attenuated pathogens is an important factor for determination of vaccine ef ficacy.Thoulouze et al.(2004)reported that rIHNV was recovered at 2weeks p.i.from one of five rainbow trout that were bath immersed with 5×104PFU of the rIHNV lacking NV ORF.However,Romero et al.(2008)described that viral bands were not detected at 3day p.i.from rainbow trout i.p.infected with the rIHNV harboring GFP ORF instead of NV ORF when analyzed by RT-PCR.Among survived yellow perch i.p.injected with105PFU of the NV-de ficient rVHSV at the end of the experiment (30d),2/6fish were positive with titers 103–104(Ammayappan et al.,2010).In the present study,we observed that the density of i.m.injected rVHSV-ΔNV-EGFP's G gene bands ampli fied by RT-PCR was gradually increased at early period p.i.,then sharply decreased with the lapse of time from the internal organs (kidney,spleen,and liver)of olive flounder reared at either 20°C or 15°C.These results suggest that the rVHSV-ΔNV-EGFPFig.3.Vaccine ef ficacy of rVHSV-ΔNV-EGFP.Olive flounder were intramuscularly immunized with rVHSV-ΔNV-EGFP (103,104,or 105PFU/fish),and boost immunized at 4weeks after the primary immunization with the same doses of the recombinant virus.(a)At 4weeks after the primary immunization half of the fish in each experimental group (2replicates)were challenged with 103PFU/fish of wild-type VHSV KJ 2008.(b)At 4weeks after the boost immunization fish in each group (2replicates)were challenged with 103PFU/fish of wild-type VHSV KJ 2008.Cumulative mortality rates were recorded daily for 30d post-challenge.(—●—,control group;—○—,105PFU/fish;—▼—,104PFU/fish;—Δ—,103PFU/fish).Fig.4.Neutralization activity against wild-type VHSV KJ 2008in sera of olive flounder (Paralichthys olivaceus )immunized by intramuscular (i.m.)injection of L-15culture medium (Control),or rVHSV-ΔNV-EGFP at three doses (103,104,or 105PFU/fish).At 4weeks post-immunization,half of the fish in each group were boost immunized with the same regime of the primary immunization.At 4weeks post-boosting,6fish in each group were bled to obtain serum for neutralization.Values are mean ±standard deviation.Different letters on the bars represent signi ficantly different at P b 0.05.42M.S.Kim,K.H.Kim /Aquaculture 314(2011)39–43has the ability to spread and replicate in the internal organs of olive flounder.Furthermore,the in vivo replication ability of the rVHSV-ΔNV-EGFP not only at15°C that is preferential for VHSV replication but also at 20°C that is preferential for induction of immune responses in olive flounder,is considered as a promising aspect for induction of efficient adaptive immune responses in oliveflounder.Because of the void of adequate therapeutics,the prevention of viral diseases in farmedfish is crucial for enhancing aquaculture productivity. Vaccination has been one of the best options for controlling infectious viral diseases.Vaccine experiments using novirhabdoviruses generated by the reverse genetics were conducted mainly on IHNV.Thoulouze et al.(2004)reported that rainbow trout immunized by immersion bath with5×104PFU of rIHNV-ΔNV-GFP/ml provided a high protection (survival rate92%)against wild-type IHNV challenge.Romero et al. (2008)also described that the cumulative mortality in the group of rainbow trout i.p.immunized with105PFU of rIHNV-ΔNV-GFP was29% when challenged with wild-type IHNV,while that in the control group was97.3%.Novoa et al.(2006)demonstrated that immunization of zebrafish with the rIHNV-Gvhsv GFP(replaced the NV gene with GFP gene and IHNV G gene with VHSV G gene)induced a high protection against wild-type IHNV challenge.In the present study,wefirstly conducted a vaccine experiment to assess the potential of the rVHSV-ΔNV-EGFP as a live attenuated vaccine candidate in oliveflounder.In our preliminary experiment,a high titer(105PFU/fish)of rVHSV-ΔNV-EGFP was needed to get a high protective efficacy by immunization in oliveflounder that were reared at15°C.Therefore,in the present study, the immunization experiment was conducted with oliveflounder reared at20–22°C and thefish were intramuscularly immunized with the rVHSV-ΔNV-EGFP at103–105PFU/fish.The survival rates of immunizedfish were dose-dependent,and were clearly enhanced by the boost immunization.The serum neutralization titers were also proportional to the survival rates of vaccinated groups,suggesting that the humoral adaptive immune responses were induced by i.m. immunization of the recombinant VHSV.The present results suggest that the rVHSV-ΔNV-EGFP has a high potential to be used as a live vaccine in oliveflounder farms,and immunization offish before low-temperature period may induce a high protection against virulent VHSV infections.In conclusion,the NV gene of VHSV plays an important role in virulence,and attenuation of VHSV by NV gene deletion is a way to develop an effective live vaccine in oliveflounder.Further researches on the delivery methods,duration of protection,and applicability to combined vaccines using recombinant VHSVs are urgently needed.AcknowledgementThis research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education,Science and Technology(2010–0015654). 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