Development and characterization of a monoclonal antibody against Taura syndrome virus

Development and characterization of a monoclonal

antibody against Taura syndrome virus

I C�t�1, B T Poulos2, R M Redman1 and D V Lightner1

1 Department of Veterinary Sciences and Microbiology, The University of Arizona, Tucson, AZ, USA

2 Department of Ecology and Evolutionary Biology, The University of Arizona, Tucson, AZ, USA

Abstract

We produced a panel of monoclonal antibodies(MAbs) from the fusion of Taura syndrome virusvariants from Belize (TSV-BZ) immunized BALB/cJ mouse spleen cells and non-immunoglobulinsecreting SP2/0 mouse myeloma cells. One anti-body, 2C4, showed strong specificity and sensitivityfor TSV in dot-blot immunoassay and immuno-histochemistry (IHC) analysis. The MAb reactedagainst native TSV-BZ, TSV variants from Sinaloa,Mexico (TSV-SI) and TSV variants from Hawaii(TSV-HI) in dot-blot immunoassay. By IHC, theantibody identified the virus in a pattern similar tothe digoxigenin-labelled TSV-cDNA probe for theTSV-BZ, TSV-HI and TSV-SI variants, but not forthe TSV variants from Venezuela (TSV-VE) andthe TSV variants from Thailand (TSV-TH). MAb2C4 did not react against other shrimp pathogensor with normal shrimp tissue. Western blot analysisshowed a strong reaction against CP2, a region ofhigh antigenic variability amongst TSV variants.This antibody has potential diagnostic applicationin detection and differentiation of certain TSVbiotypes.

Keywords: immunodiagnosis, monoclonal antibody,Taura syndrome virus.

Introduction

In 1992, Jimenez described Taura syndrome (TS)in Ecuador (Jimenez 1992). Since that year, TS hasbecome a major threat to the shrimp farming

industry worldwide causing outbreaks in mostshrimp farming areas of the Americas (Hasson,Lightner, Mari, Bonami, Poulos, Mohney, Redman& Brock 1999a) and South-East Asia (Tu, Huang,Chuang, Hsu, Kuo, Li, Hsu, Li & Lin 1999;Nielsen, Sang-Oum, Cheevadhanarak & Flegel2005). The causative agent of the disease is anon-enveloped, single-stranded positive sense RNAvirus member of the Discistroviridae family: TSvirus (TSV) (Mari, Poulos, Lightner & Bonami2002; Mayo & Ball 2006). The fully sequencedgenome is 10 205 nucleotides in length andencodes two open-reading frames (ORFs). The firstORF encodes for three genes: a helicase, a proteaseand a RNA-dependent RNA polymerase. Thecapsid consists of three major polypeptides encodedin the second ORF: CP1 (40 kDa), CP2 (55 kDa)and CP3 (24 kDa), and a minor protein of 58 kDa(Mari et al. 2002).

The disease has three overlapping phases: acute,transitional and chronic (Hasson, Lightner, Moh-ney, Redman, Poulos & White 1999b). In the acutephase, shrimp are lethargic and sometimes dieabruptly. This phase is followed by a transitionalphase, with shrimp displaying characteristic melan-ized lesions distributed randomly on the cuticle.Survivors of this second phase enter the chronicstage of the disease during which they do notdisplay any outward signs of infection. On histo-logical examination, �buckshot� lesions are observedin the cuticle of the gills, stomach and epidermisduring the acute and transitional phases (Hassonet al.1999b). Animals in chronic phase presentlymphoid organ spheroids and the absence of otherlesions (Hasson, Lightner, Mohney, Redman &White 1999c).

Treatment measures against shrimp viruses arelimited and efforts have been put toward the raising

Journal of Fish Diseases 2009, 32, 989–996 doi:10.1111/j.1365-2761.2009.01082.x

Correspondence I Cote, Department of Veterinary Sciences

and Microbiology, The University of Arizona, Tucson, AZ 85721,

USA

(e-mail: [email protected])

989

� 2009 The Authors.

Journal compilation

� 2009

Blackwell Publishing Ltd

of TSV-resistant animals. Resistant lines havebeen successfully developed and their use haslimited the impact of the disease. However, viralvariants have emerged to which resistant shrimplines might be susceptible (Argue, Arce, Lotz &Moss 2002; Erickson, Zarain-Herzberg & Lightner2002).

Reverse transcriptase polymerase chain reaction(RT-PCR) has been developed for diagnosis of TSVinfection (Nunan, Poulos & Lightner 1998). Geneprobes are also available for the detection of the virusin fixed tissue sections (Mari, Bonami & Lightner1998). These RNA-based detection methods requirepreservation of the relatively fragile viral RNA(Hasson, Hasson, Aubert, Redman & Lightner1997). Compared with RNA-based methods, anti-body-based methods recognizing the more stablecapsid proteins are advantageous in field situationsbecause they require less technical expertise and notoxic chemicals (Sithigorngul, Rukpratanporn,Pecharaburanin, Longyant, Chaivisuthangkura &Sithigorngul 2006).

Mutation rates are higher in RNA viruses thanin DNA viruses because of the lack of fidelity andproofreading activity during replication. Thefrequency of errors during RNA genome replica-tion can be as high as 1 misincorporation per103–104 nucleotides polymerised (Holland, Spin-dler, Horodyski, Grabau, Nichol & Vandepol1982). Variants of TSV are named with the placeand date of first isolation: TSV-HI94, TSV-SI98,TSV-BZ02, TSV-VE05 and TSV-TH05 (Erick-son et al. 2002; Erickson, Poulos, Tang, Bradley-Dunlop & Lightner 2005; Srisuvan, Noble,Schofield & Lightner 2006; Cote, Navarro, Tang,Noble & Lightner 2008). Hereafter these variantsare referred to as TSV-HI, TSV-SI, TSV-BZ,TSV-VE and TSV-TH. The genetic zone of highvariability is in the gene encoding CP2 whereregions are sequenced (Tang & Lightner 2005).TSV serotypes have been tentatively attributedwith regard to the reaction with the currentlyavailable monoclonal antibodies (MAbs) for TSVCP2, 1A1 (Poulos, Kibler, Bradley-Dunlop,Mohney & Lightner 1999; Erickson, Poulos,Bradley-Dunlop, White-Noble & Lightner 2003).MAb 1A1 recognizes TSV-HI, TSV-TH andTSV-VE (native form only) and no other variantsof TSV (Poulos et al. 1999; Cote et al. 2008).Here we report on the development of a MAbthat recognizes TSV-BZ, TSV-HI and TSV-SI,but not TSV-TH and TSV-VE.

Materials and methods

Experimental shrimp

Oceanic Institute�s specific pathogen-free (SPF)breeding programme in Hawaii provided SPF Konastock Penaeus vannamei (Holthuis 1980; Wyban,Swingle, Sweene & Pruder 1992). The shrimp werecultured from post-larvae to the juvenile stage usedin this study at the Aquaculture Pathology Labo-ratory of the University of Arizona as described inWhite, Schofield, Poulos & Lightner (2002).

Antigens

For the production of antigens, Kona StockP. vannamei were injected with 50 lm of a 1:10homogenate of TSV-BZ infected shrimp tissue in2% saline. Shrimp were maintained at a density of10 per 90 L tank. All tanks were equipped with abiofilter. Moribund and dead shrimp were removedfrom the tank, usually between 3 and 5 days afterthe injection, and frozen at )70 �C until used forviral purification. Viral purification was performedas described by Bonami, Hasson, Mari, Poulos &Lightner (1997) with minor modifications. Follow-ing the sucrose gradient the fractions were dilutedin 10 mm Tris–400 mm NaCl (TN) 1:2 andcentrifuged for 3 h at 286 000 g. The resultingpellet was suspended in 1 mL of filter-sterilized TNwith 1 lL (0.1%) of Triton X-100 and incubatedfor 1 h on ice on a rocking apparatus [modifiedfrom Tsukamoto, Obata & Hihara (1990)]. Thesolution was thereafter layered onto a 15–45%caesium chloride gradient and centrifuged for 13 hat 208 500 g. Fractions associated with the bandwere collected, pooled and diluted 1:2 in TN andcentrifuged at 286 000 g for 3 h. The supernatantwas discarded and the pellet was suspended in filter-sterilized TN. The virus preparation was negativelystained with 2% phosphotungstic acid and exam-ined for purity analysis with a JEM-100CXII (TEMJOEL Co, Ltd) transmission electron microscope(TEM). The same procedure was used to purifyTSV-HI, TSV-SI and TSV-VE using archivedshrimp or tissue homogenate. A triton wash wasnot performed on these later viruses. The genomecopy number in all four viral preparations wasdetermined with a real-time RT-PCR TaqMan assayas described by Tang, Wang & Lightner (2004).

The CP2 region of the purified viruses wasamplified using the primers 55P1 and 55P2 asdescribed by Tang & Lightner (2005), using 1 lL

990


Journal compilation

� 2009


Journal of Fish Diseases 2009, 32, 989–996 I Cote et al. Monoclonal antibody against Taura syndrome virus

of the purified virus solution in the PCR reaction. A10 lL aliquot of the amplified product was analysedin a 1.5% agarose gel containing ethidium bromide.The amplified fragment was cleaned using a QIA-Quick PCR purification kit (Quiagen) according tothe manufacturer�s recommendations. The fragmentswere sequenced with an ABI Prism 377 automatedDNA sequencer by the sequencing facility of theUniversity of Arizona. The sequence obtained wasblasted against TSV sequences in the NIH database(http://www.ncbi.nlm.nih.gov) for homology.

Haemolymph was collected from shrimp infectedwith yellow head virus (YHV) or white spotsyndrome virus (WSSV), as well as from SPFPenaeus monodon and P. vannamei. The haemol-ymph was frozen at )70 �C until used in dot-blotimmunoassay analysis.

Immunization

Five-week-old Balb/c mice were obtained fromJackson Laboratory (Bar Harbor) and kept at theCentral Animal Care Facility of the University ofArizona for at least 2 weeks prior to the immuniza-tion procedure. All mice manipulations were per-formed in accordance with the recommendationsfrom the Institutional Animal Care and Use Com-mittee of the University of Arizona. For the immu-nization of the mice, a mixed viral solution of nativeand denatured (boiled for 5 min) in a ratio of 1:1 wasprepared using purified TSV-BZ. The viral antigensolution was diluted with Titermax gold adjuvant(Sigma) prepared following the manufacturer�s rec-ommendations. Mice were injected with 100 lL intwo sites on the flank (50 lL each site). This wasfollowed by a booster injection 3 weeks later. Anintraperitoneal injection booster without adjuvantwas given exactly 72 h prior to the fusion.

Fusion

The procedure for fusion was carried out using thestandard polyethylene glycol-mediated proceduredescribed by Gefter, Margulies & Scharff (1977).Spleen cells from the immunized mice were fusedwith approximately 1E7 SP2 myeloma cells pre-treated with 8-azaguanine (Sigma). The resultingcell solution was plated in 24-well Costar plates(Corning) with RPMI-conditioned culture mediumwith 20% heat inactivated foetal calf serum and 5%Briclone (QED Bioscience) for 24 h. After 24 h,culture medium with 2· hypoxanthine–aminopter-

ine–thymidine (HAT; Sigma) was added for a finalconcentration of 1· HAT. The cells were fed asdeemed necessary by their appearance and the pHof the media. After 15 days, the cells were fedhypoxanthine-thymidine (HT; Sigma) media with-out aminopterine. At all stages of culture, cells weremaintained at 37 �C in a 5% CO2 moisturizedatmosphere. The water bath in the incubator waschanged monthly and contained 0.5% Aquaclean(WAK-Chemie). The cells were maintained underconditions of strict sterility and no antibiotics wereadded during the culture process. The resultanthybridoma cell cultures were tested for the produc-tion of antibodies specific to TSV by dot-blotimmunoassay of the supernatant on purified TSVand SPF shrimp haemolymph.

The dot-blot immunoassay was performed asdescribed by Poulos et al. (1999) with minormodifications. Briefly, MAHAN 45 plates (Milli-pore) were dotted with 1 lL of purified virus and1 lL of SPF haemolymph and allowed to dry. Theywere blocked at 37 �C for 30 min with 150 lL of10% normal goat serum and 10% fat-free pow-dered milk in phosphate buffer solution (PBS). Thewells were then reacted with 100 lL of hybridomasupernatant fluid for 30 min (sometimes incuba-tion times up to 4 h were used) at 37 �C; mice sera(PAbs) diluted 1:100 in PBS was used as a positivecontrol. The wells were washed three times withPBS and reacted with 100 lL of a 1:1000 dilutionof phosphatase-labelled goat-anti-mouse F(ab¢)2

fragments (Kierkegaard & Perry) for 30 min at37 �C. Wells were washed three times with PBSand the reactions were visualized by developmentwith nitroblue tetrazolium and bromo-chloro-in-doyl phosphate for 30–45 min in the dark. Wellsspecific to TSV were cloned four times by limitingdilution. Cell numbers were expanded in 25 cm2

flasks and frozen at all stages of culture.

Dot-blot immunoassay

Dot-blot immunoassay was performed as previouslydescribed on haemolymph infected with YHV,WSSV, SPF P. vannamei, SPF P. monodon as wellas purified TSV-BZ, TSV-HI, TSV-SI and TSV-VE. TSV-TH was not tested.

Immunohistochemistry and in situ hybridization

Cases from our diagnostic database that werefound positive for TSV by standard histology

991


Journal compilation

� 2009



were chosen to perform immunohistochemistry(IHC) as described by Poulos, Pantoja, Bradley-Dunlop, Aguilar & Lightner (2001) with modi-fications. Briefly, paraffin-embedded samples werecut in 4 lm sections, heated at 65 �C for 30 minand rehydrated. The slides were washed for 5 minin PBS and blocked for 30 min in a humidchamber at 37 �C using blocking buffer[PBS + 2% dry milk + 10% normal goat serum(NGS)]. The slides were reacted with 500 lL ofhybridoma supernatant fluid from cell culture thatwas positive for TSV-BZ in immunodoblot. Theslides were washed three times (2 min each wash)with PBS and incubated with 500 lL of 1:1000goat-anti-mouse antibody conjugated with alkalinephosphatase at 37 �C for 30 min. The slides werewashed three more times and incubated withdeveloping solution for 1 h at room temperaturein the dark. The slides were counter-stained withBismarck brown, dehydrated and coverslippedusing permount. In situ hybridization (ISH) wasperformed on sections from the same tissuesample with the P15-Q1 probe using the proce-dure described by Mari et al. (1998).

Immunohistochemistry was also performed onsamples infected with hepatopancreatitis parvovirus(HPV), infectious hypodermal and haemopoieticnecrosis (IHHNV), infectious myonecrosis virus(IMNV), YHV, WSSV, Spiroplasma penaei andSPF P. vannamei.

SDS-PAGE and Western blot analysis

Taura syndrome virus preparations (HI, SI andBZ) were denatured in Laemmli buffer (Laemmli1970) with 10% urea and separated using a 12%resolving polyacrylamide gel (Bonami et al. 1997)at 25 mAmp for 100 min. A prestained kaleido-scope polypeptide standard (Bio-Rad) and apremixed molecular mass marker (Roche) wererun alongside the virus for reference. Gels wereeither stained with 0.1% Coomassie blue (Wilson1983) or transferred to nitrocellulose membranefor Western blot analysis as described by Pouloset al. (1999).

The membranes were dried overnight at roomtemperature. The membranes were reacted withundiluted supernatant or mouse PAb diluted1:1000 in PBS with Tween-20 + 10% NGS.Antibody incubations were performed in bags for1 h on a rocking apparatus. The reactions werevisualized as in the dot-blot immunoassay.

Isotype determination

The isotype of the antibody was determined usingthe ImmunoPure Monoclonal Antibody IsotypingKit II (Pierce) following the antigen-independentELISA procedure as described by the manufac-turer.

Results

Antigens

With TEM the purified virus preparations ofTSV-VE, TSV-BZ and TSV-SI were found tocontain only one type of virus particle that wereconsistent with TSV as previously illustrated inHasson, Lightner, Poulos, Redman, White, Brock& Bonami (1995). The viral load of the TSV-BZantigens used for the mouse immunization was6.84E9 viral RNA copies lL)1 as determined byreal-time quantitative RT-PCR (qRT-PCR). Titresof all viruses were obtained and these were dilutedin PBS to obtain identical concentrations for usein the dot-blot immunoassay. The TSV-VE prep-aration was not as concentrated; therefore, we usedTSV-VE for the dot-blot immunoassay but not forthe Western blot assay, as the resolution onsodium dodecyl–sulfate polyacrylamide gel elec-trophoresis (SDS-PAGE) was very poor.

Monoclonal antibody production

The selected clone, 2C4, was stable in culture andproduced antibodies even after being subcloned 25times over a period of 2 months. This clone wasfrozen and retrieved without loss of antibodyproduction. None of the other clones developedproved to be specific to TSV and highly stable.Some clones showed faint positive reactions toTSV, but these disappeared following cloning.

Determination of isotype

The MAb 2C4 was determined to be of the IgG1jisotype using the ImmunoPure Monoclonal Anti-body Isotyping Kit II (Pierce).

Antigen specificity of MAbs

We tested MAb 2C4 by dot-blot immunoassayagainst TSV (four variants) and by IHC (with fiveTSV variants). By dot-blot immunoassay the antibody

992


Journal compilation

� 2009



reacted against the following variants: TSV-BZ,TSV-HI and TSV-SI, but not against TSV-VE(Fig. 1). By IHC the antibody reacted with shrimptissue infected with TSV-BZ, TSV-HI and TSV-SIbut not with shrimp tissue infected with TSV-VEand TSV-TH (Table 1). For IHC, samples fromboth the acute and chronic phases were included inthe analysis. All TSV-infected tissue sections exam-ined were positive by ISH with a TSV specificcDNA probe. It was observed that, in general, IHCwith 2C4 did not give as strong results as ISH (seeFig. 2 for representative ISH and IHC images).However, the antibody was sensitive as all shrimpinfected with TSV-BZ, TSV-HI or TSV-SI thatwere positive by ISH were also detected by IHC.

The 2C4 antibody was highly specific for TSV. Itdid not react by dot-blot immunoassay with YHV,WSSV or SPF haemolymph. It was negative byIHC against all other shrimp pathogens tested:HPV, IHHNV, IMNV, YHV, WSSV and Spiropl-asma penaei (data not shown).

SDS-PAGE

By SDS-PAGE, the protein profile of the purifiedvirus (TSV-BZ, TSV-HI and TSV-SI) containedthree major proteins of 55, 40 and 24 kDa. This isconsistent with Bonami et al. (1997). The TSV-HIpreparation was more concentrated and showedstronger bands (Fig. 3).

Figure 1 From left to right: dotted Taura syndrome virus variants from Belize (TSV-BZ), Hawaii (TSV-HI), Sinaloa, Mexico

(TSV-SI), and Venezuela (TSV-VE) reacted with monoclonal antibody 2C4 followed by the viruses in the same order reacted with

PAbs. A positive reaction is indicated by a dark spot on the membrane.

Table 1 Sensitivity of the anti-Taura syndrome virus variants (TSV) MAb 2C4 as determined by immunohistochemistry (IHC) on

fixed tissue sections and compared with in situ hybridization (ISH) with TSV digoxigenin-labelled cDNA probe and histological findings

of the same sample

Sample no. Variant Days p.i. ISH IHC Histological findings

1 BZ 15 + + LOS4

2 BZ 1 + + Buckshota G1–4b

3 BZ 2 + + LOS3, buckshot G2–3

4 BZ 3 + + LOS2, buckshot G2–4

5 BZ 3 + + Buckshot G2–4, no LO

6 HI 15 + + LOS3–4

7 HI 5 + + Buckshot G1–2

8 HI 4 + + LOS2, buckshot G2–3

9 HI 4 + + LOS2, buckshot G1

10 HI 9 + + LOS3, buckshot G2

11 HI 7 + + LOS2, buckshot G2–4

12 SI 2 + + LOS2, buckshot G3–4

13 SI 15 + + LOS3

14 SI 15 + + LOS4

15 SI 2 + + Buckshot G1–2

16 SI 3 + + Buckshot G1–2,

granuloma G4 LO

17 TH 3 + ) Buckshot G2

18 TH 3 + ) Buckshot G2–3

19 TH 4 + ) LOS1–2

20 TH 4 + ) LOS2, buckshot G2–3

21 TH 15 + ) LOS3

22 VE 6 + ) LOS2, buckshot G2–3


24 VE 2 + ) LOS1, buckshot G1


26 VE 14 + ) LOS3

Monoclonal antibody (MAb) 2C4 failed to detect two variants of TSV (Thailand and Venezuela) by this method.

LOS, lymphoid organ spheroid; Taura syndrome virus variants from: BZ, Belize; HI, Hawaii; SI, Sinaloa, Mexico; VE, Venezuela; TH, Thailand;

+, positive; ), negative.aBuckshot, acute TSV lesions.bG1 to G4, severity grade of infection/lesion according to the scale described in Hasson et al. (1995).

993


Journal compilation

� 2009



Western blot analysis

Western blot was performed with the BZ, HI andSI variants of TSV. MAb 2C4 recognized CP2 onall three variants. A small faint band was observedunder the TSV-HI variant CP2. PAbs reactedstrongly with CP2 of all variants tested (data notshown). The absence of strong reactions with theother bands CP1 and CP3 is consistent with Pouloset al. (1999) who observed a very weak reactionwith CP1 and no reaction with CP3 using PAbsagainst TSV-HI.

Discussion

The purpose of this study was to develop a panel ofhighly specific MAbs that could be used in acocktail recognizing all strains of TSV and individ-ually recognize specific TSV serotypes. Only onemouse hybridoma cell line producing MAbs against

TSV was obtained. The antibody produced, desig-nated MAb 2C4, was of the IgG1j isotype. Itconsistently demonstrated highly specific reactionsin all formats (IHC, dot-blot immunoassay andWestern blot) tested.

Using IHC, no positive reaction was found inhealthy shrimp tissue, tissue infected with otherpathogens or non-target tissues in TSV-infectedshrimp. There was no reaction with any of theTSV-TH and TSV-VE infected tissue sectionsexamined by IHC. These tissue sections were,however, positive for TSV by ISH, indicating thatthe absence of reaction with MAb 2C4 was notbecause of the absence of the virus.

The antibody reacted strongly with the bandassociated with CP2 in Western blot as well as witha band of slightly lower molecular weight for TSV-HI. The significance of this band is not known. Itcould represent a cleavage product of CP2 thatconserves some of the same protein determinants.Some viruses, such as Acyrthosiphon pisum virus,present proteolytic break-down of some of theirpolypeptides resulting in capsid protein of varyinglengths with identical series of amino acids (van derWilk, Dullemands, Verbeek & Van den Heuvel1997). It is not known why this band was present inTSV-HI but not present in TSV-BZ or TSV-SI.The relative quantity of the TSV-HI virus particlesused may account for this band being visualized forTSV-HI but not in the other two preparations.

(a)

(b)

Figure 2 Immunohistochemistry and in situ hybridization

(ISH) reactions in Penaeus vannamei tissues 7 days post-infection

with Taura syndrome virus variant from Hawaii (TSV-HI).

(a) ISH with a digoxigenin-labelled TSV-cDNA probe.

(b) IHC with monoclonal antibody 2C4 (·100).

Figure 3 Sodium dodecyl sulfate polyacrylamide gel electro-

phoresis (SDS-PAGE) of proteins from purified Taura syndrome

virus (TVS) isolates stained with Coomassie Blue. Lane 1, Taura

syndrome virus variant from Belize (TSV-BZ). Lane 2,

Taura syndrome virus variant from Hawaii (TSV-HI). Lane 3,

Taura syndrome virus variant from Sinaoa, Mexico (TSV-SI).

Lane 4, molecular weight marker; molecular masses of the

proteins (kDa).

994


Journal compilation

� 2009



CP2 is a region of high genetic variabilityamongst TSV variants. MAbs recognize epitopessometimes as small as four amino-acids (Somins-kaya, Pushko, Dreilina, Kozlovaskaya & Pumpen1992). It could be hypothesized that these wereconserved for BZ, HI and SI but not for VE andTH. Similar to MAb 2C4, MAb 1A1 reacts againstCP2. This opens interesting avenues for classifyingstrains of TSV into serotypes as CP2 is the region ofhighest genetic variation in the TSV capsid. Thedetection ability of the currently available cocktailof MAbs (1A1 and 2C4) includes TSV-TH,TSV-HI, TSV-SI, TSV-BZ and the native formof TSV-VE. This covers most variants currentlyencountered in the Americas and Asia and could beused as a good first line diagnostic test for TS.Other authors have reported the production ofMAbs against another capsid protein of TSV: CP3.However, these MAbs have not been tested againstall the variants examined in this study (Longyant,Poyoi, Chaivisuthangkura, Tejangkura, Sithigorn-gul, Sithigorngul & Rukpratanporn 2008).

Despite numerous fusions and the production ofseveral healthy clones, only one MAb was producedin the present study. Poulos et al. (1999) havereported similar problems, in which three clonesthat reacted with TSV were developed, but of theseonly one consistently presented strong reactions.This success rate is lower compared with otheraquatic pathogens whether bacterial such as Pisci-rickettsia salmonis (Jamett, Aguayo, Miquel, Muller,Arriagada, Becker, Valenzuela & Burzio 2001) orviral such as WSSV (Shih, Wang, Tan & Chen2001). In these cases, panels of 28 and 20,respectively, MAbs or positive hybridoma cloneswere produced.

Many of the first hybridoma clones producedshowed reactivity with shrimp haemolymph. This issurprising considering that the virus was extensivelypurified and taken from a very sharp peak on thegradient. However, as stated by Berry (2005), wholepathogen immunogen preparations are inherentlycontaminated unless they can be grown in pureform. Since TSV has not been cultivated in vitro,we tried to address this issue by washing the viralpreparation with Triton-X. Shih et al. (2001) havedescribed a MAb produced following injection ofpurified WSSV that bound specifically to twoshrimp proteins and reacted to healthy and non-target infected tissues.

These results strongly suggest that MAb 2C4 isuseful for the diagnosis of TSV-HI, TSV-SI and

TSV-BZ infection and provides useful informationfor the classification of TSV variants. It presents astep towards improved rapid detection methods forTSV.

Acknowledgements

The principal author is most thankful to D.Bradley-Dunlop (Department of Immunobiology,University of Arizona) for technical advice. Grantsupport for this study was from the United StatesMarine Shrimp Farming Consortium under GrantNo. 2004-38808-02142 from the Cooperative StateResearch, Education and Extension Service, U.S.Department of Agriculture.

References

Argue B.J., Arce S.M., Lotz J.M. & Moss S.M. (2002) Selective

breeding of Pacific white shrimp (Litopeaneus vannamei) for

growth and resistance to Taura syndrome virus. Aquaculture204, 447–460.

Berry J.D. (2005) Rational monoclonal antibody development to

emerging pathogens, biothreat agents and agents of foreign

animal disease: the antigen scale. The Veterinary Journal 170,

193–211.

Bonami J.R., Hasson K.W., Mari J., Poulos B.T. & Lightner

D.V. (1997) Taura syndrome of marine penaeid shrimp:

characterization of the viral agent. Journal of General Virology78, 313–319.

Cote I., Navarro S., Tang K.F.J., Noble B. & Lightner D.V.

(2008) Taura syndrome virus from Venezuela is a new genetic

variant. Aquaculture 284, 62–67.

Erickson H.S., Zarain-Herzberg M. & Lightner D.V. (2002)

Detection of Taura syndrome virus (TSV) strain differences

using selected diagnostic methods: diagnostic implications in

penaeid shrimp. Diseases of Aquatic Organisms 52, 1–10.

Erickson H.S., Poulos B.T., Bradley-Dunlop D., White-Noble

B. & Lightner D.V. (2003) Diagnostic profile of Belize Taura

syndrome virus. Global Aquaculture Advocate 6, 88–89.

Erickson H.S., Poulos B.T., Tang K.F.J., Bradley-Dunlop D. &

Lightner D.V. (2005) Taura syndrome virus from Belize

represents a unique variant. Diseases of Aquatic Organisms 64,

91–98.

Gefter M.L., Margulies D.H. & Scharff M.D. (1977) A simple

method for polyethylene glycol-promoted hybridization of

mouse myeloma cells. Somatic Cell Genetics 3, 231–236.

Hasson K.W., Lightner D.V., Poulos B.T., Redman R.M.,

White B.L., Brock J.A. & Bonami J.R. (1995) Taura syn-

drome in Penaeus vannamei: demonstration of a viral etiology.

Diseases of Aquatic Organisms 23, 115–126.

Hasson K.W., Hasson J., Aubert H., Redman R.M. & Lightner

D.V. (1997) A new RNA-friendly fixative for the preservation

of penaeid shrimp samples for virological detection using

cDNA genomic probes. Journal of Virological Methods 66,

227–236.

995


Journal compilation

� 2009



Hasson K.W., Lightner D.V., Mari J., Bonami J.R., Poulos B.T.,

Mohney L.L., Redman R.M. & Brock J.A. (1999a) The geo-

graphic distribution of Taura syndrome virus (TSV) in the

Americas: determination by histopathology and in situ hybridiza-

tion using TSV-specific cDNA probes. Aquaculture 171, 13–26.

Hasson K.W., Lightner D.V., Mohney L.L., Redman R.M.,

Poulos B.T. & White B.M. (1999b) Taura syndrome virus

(TSV) lesion development and the disease cycle in the Pacific

white shrimp Penaeus vannamei. Diseases of Aquatic Organisms36, 81–93.

Hasson K.W., Lightner D.V., Mohney L.L., Redman R.M. &

White B.M. (1999c) Role of lymphoid organ spheroids in

chronic Taura syndrome virus (TSV) infections in Penaeusvannamei. Diseases of Aquatic Organisms 38, 93–105.

Holland J., Spindler K., Horodyski F., Grabau E., Nichol S. &

Vandepol S. (1982) Rapid evolution of RNA genomes. Science215, 1577–1585.

Holthuis L.B. (1980) Shrimp and prawns of the world. FAOSpecies Catalogue, FAO Fisheries Synopsis 125. Food and

Agriculture Organization of the United Nations, Rome.

Jamett A., Aguayo J., Miquel A., Muller I., Arriagada R., Becker

M.I., Valenzuela P. & Burzio L.O. (2001) Characteristics of

monoclonal antibodies against Piscirickettsia salmonis. Journalof Fish Diseases 24, 205–215.

Jimenez R. (1992) Sindrome de Taura (resumen) Acuacultura

del Ecuador. In Rev Esp Camara Nacional Acuacultura,Guayaquil, Ecuador, December 1, pp. 1–16 (in Spanish).

Laemmli U.K. (1970) Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature 227, 680–

685.

Longyant S., Poyoi P., Chaivisuthangkura P., Tejangkura T.,

Sithigorngul W., Sithigorngul P. & Rukpratanporn S. (2008)

Specific monoclonal antibodies raised against Taura syndrome

virus (TSV) capsid protein VP3 detect TSV in single and dual

infections with white spot syndrome virus (WSSV). Diseases ofAquatic Organisms 79, 75–81.

Mari J., Bonami J.R. & Lightner D.V. (1998) Taura syndrome

of penaeid shrimp: cloning of viral genome fragments and

development of specific gene probes. Diseases of AquaticOrganisms 33, 11–17.

Mari J., Poulos B.T., Lightner D.V. & Bonami J.R. (2002)

Shrimp Taura syndrome virus: genomic characterization and

similarity with members of the genus Cricket paralysis-like

viruses. Journal of General Virology 83, 915–926.

Mayo M.A. & Ball L.A. (2006) ICTV in San Francisco: a report

from the plenary session. Archives of Virology 151, 413–422.

Nielsen L., Sang-Oum W., Cheevadhanarak S. & Flegel T.W.

(2005) Taura syndrome virus (TSV) in Thailand and its

relationship to TSV in China and the Americas. Diseases ofAquatic Organisms 63, 101–106.

Nunan L.M., Poulos B.T. & Lightner D.V. (1998) Reverse

transcription polymerase chain reaction (RT-PCR) used for

the detection of Taura syndrome virus (TSV) in experimen-

tally infected shrimp. Diseases of Aquatic Organisms 34, 87–91.

Poulos B.T., Kibler R., Bradley-Dunlop D., Mohney L.L. &

Lightner D.V. (1999) Production and use of antibodies for the

detection of Taura syndrome virus in penaeid shrimp. Diseasesof Aquatic Organisms 37, 99–106.

Poulos B.T., Pantoja C.R., Bradley-Dunlop D., Aguilar J. &

Lightner D.V. (2001) Development and application of

monoclonal antibodies for the detection of white spot syn-

drome virus of penaeid shrimp. Diseases of Aquatic Organisms47, 13–23.

Shih H.H., Wang C.S., Tan L.F. & Chen S.N. (2001) Char-

acterization and application of monoclonal antibodies against

white spot syndrome virus. Journal of Fish Diseases 24, 143–

150.

Sithigorngul W., Rukpratanporn S., Pecharaburanin N.,

Longyant S., Chaivisuthangkura P. & Sithigorngul P. (2006)

A simple and rapid immunochromatographic test strip for

detection of white spot syndrome virus (WSSV) of shrimp.


Sominskaya I., Pushko P., Dreilina D., Kozlovaskaya T. &

Pumpen P. (1992) Determination of the minimal length of

preS1 epitope recognized by a monoclonal antibody which

inhibits attachment of Hepatitis B virus to hepatocytes.

Medical Microbiology and Immunology 181, 215–226.

Srisuvan T., Noble B.L., Schofield P.J. & Lightner D.V. (2006)

Comparison of four Taura syndrome virus (TSV) isolates in

oral challenge studies with Litopenaeus vannamei unselected or

selected for resistance to TSV. Diseases of Aquatic Organisms71, 1–10.

Tang K.F.J. & Lightner D.V. (2005) Phylogenetic analysis of

Taura syndrome virus isolates collected between 1993 and

2004 and virulence comparison between two isolates repre-

senting different genetic variants. Virus Research 112, 69–76.

Tang K.F.J., Wang J. & Lightner D.V. (2004) Quantitation of

Taura syndrome virus by real-time RT-PCR with a TaqMan

assay. Journal of Virological Methods 115, 109–114.

Tsukamoto K., Obata H. & Hihara H. (1990) Improved prep-

aration of ELISA antigen of infectious bursal disease virus.

Avian Diseases 34, 209–213.

Tu C., Huang H.T., Chuang S.H., Hsu J.P., Kuo S.T., Li N.J.,

Hsu T.L., Li M.C. & Lin S.Y. (1999) Taura syndrome in

Pacific white shrimp Penaeus vannamei cultured in Taiwan.


White B.L., Schofield P.J., Poulos B.T. & Lightner D.V. (2002)

A laboratory challenge method for estimating Taura syndrome

virus resistance in selected lines of Pacific white shrimp

Litopenaeus vannamei. Journal of the World Aquaculture Society33, 341–348.

van der Wilk F., Dullemands A.M., Verbeek M. & Van den

Heuvel J.F.J.M. (1997) Nucleotide sequence and genomic

organization of Acyrthosiphon Pisum virus. Virology 238, 352–

362.

Wilson C.M. (1983) Staining of proteins on gels: comparison of

dyes and procedures. Methods in Enzymology 91, 236–247.

Wyban J.A., Swingle J.S., Sweene J.N. & Pruder G.D. (1992)

Development and commercial performance of high health

shrimp using specific pathogen free (SPF) broodstock Penaeusvannamei. In: Proceedings of the Special Session on ShrimpFarming (ed. by J. Wyban), pp. 254–259. World Aquaculture

Society, Baton Rouge, LA.

Received: 29 December 2008Revision received: 2 April 2009Accepted: 8 April 2009

996


Journal compilation

� 2009



Documents

Development and characterization of a monoclonal antibody against Taura syndrome virus