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Journal of Virological Methods 184 (2012) 121– 124

Contents lists available at SciVerse ScienceDirect

Journal of Virological Methods

jou rn al h om epage: www.elsev ier .com/ locate / jv i romet

hort communication

new method for quantifying white spot syndrome virus:xperimental challenge dose using TaqMan real-time PCR assay

ei Zhua,∗, Haizhi Quanb

College of Animal Science and Technology, Zhejiang Agriculture and Forestry University, Lin’an 311300, ChinaInstitute of Feed Science, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China

rticle history:eceived 29 November 2011eceived in revised form 19 May 2012ccepted 24 May 2012vailable online 1 June 2012

a b s t r a c t

White spot syndrome virus (WSSV) is an important pathogen in shrimp aquaculture. The susceptibilityof crayfish (Procambarus clarkii) was assessed by means of serial dilutions of a solution containing WSSV.A TaqMan real-time PCR was used to quantify the WSSV challenge dose in P. clarkii. The results showedthat WSSV copies could be detected at concentrations from 1.365 × 104 to 1.129 × 109 copies/�l. Theviral infectivity (LD50), measured as the mortality of infected crayfish, indicated 60% mortality in the 105

eywords:aqMan real-time PCRhite spot syndrome virus

rocambarus clarkiihallenge dose

dilution group (1.524 × 105 copies/�l). TaqMan real-time PCR represents a novel standard method, basedon the by quantitation of WSSV copies, for determining the appropriate concentration of WSSV for usein infection experiments.

© 2012 Elsevier B.V. All rights reserved.

xperimental methods

. Introduction

White spot syndrome virus (WSSV), which was first discoveredn Taiwan in 1992, has caused mass mortalities and devastatingroduction losses to shrimp farming over many areas (Huang et al.,995; Wang et al., 1995; Wongteerasupaya et al., 1995; Lightner,996). WSSV possesses a large DNA genome of about 300 kb, andenome analysis has shown that WSSV may be the sole member ofhe monotypic family Nimaviridae, genus Whispovirus (Van-Hultent al., 2001; Yang et al., 2001; Vlak et al., 2005). WSSV is known tonfect many crustacean species, including crayfish (Hossain et al.,001; Lo et al., 1996). Baumgartner et al. (2009) recently found thatoth farmed and wild Procambarus clarkii in Louisiana (USA) wereatural hosts for WSSV. Crayfish, such as Cherax quadricarinatusave also been used as experimental hosts for WSSV (Shi et al.,000).

A new real-time PCR technique, using TaqMan probe fluores-ence, has been used recently to quantify the copy number of aarticular target segment of nucleic acid, based on monitoring the

ncrease in fluorescence. In previous studies, TaqMan real-time PCRas used to successfully quantify WSSV in various shrimp species,

uch as Penaeus monodon, Litopenaeus vannamei and Fennerope-aeus chinensis (Jang et al., 2009; Fouzi et al., 2010; Meng et al.,010). In this investigation, a quantitative assay, involving a viral

∗ Corresponding author. Tel.: +86 571 63740815; fax: +86 571 63740815.E-mail address: zhufei1998@yahoo.cn (F. Zhu).

166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jviromet.2012.05.026

titration of WSSV in P. clarkii, was described and determine preciselethal dosages for use in challenge experiments.

2. Materials and methods

2.1. Crayfish

Crayfish (P. clarkii) of approximately 20 g were reared at25 ± 1 ◦C. They were kept in tanks with sand-filtered and ozone-treated water and fed with commercial pellet feed at 5% of bodyweight per day. The appendages from individuals selected at ran-dom were subjected to PCR assays to ensure that the crayfish wereWSSV-free prior to the experimental challenge.

2.2. WSSV stock

White spot syndrome virus-infected shrimp, F. chinensis, werecollected from shrimp farms located near Ningbo, China. Ten gramsof infected tissues (from the gills and the tail muscle) were homog-enized in 500 mL TNE buffer (50 mM Tris–HCl, 400 mM NaCl, 5 mMEDTA, pH 7.5) containing a combination of protease inhibitors —(1 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM benzami-dine, and 1 mM Na2S2O5) — and then centrifuged at 10,000 × g

for 10 min at 4 ◦C. After filtration through a nylon net (400 mesh),the homogenate was centrifuged at 6000 × g for 25 min at 4 ◦C andfiltrated using a Millipore filter (pore size 0.45 mm). The filtraterepresented the original viral fluid used for further challenge tests.

122 F. Zhu, H. Quan / Journal of Virologica

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ig. 1. The standard curve of TaqMan Real-time PCR. R: correlation coefficient; M:lope; B: intercept; Efficiency: PCR amplification efficiency.

.3. WSSV detection by PCR and quantitative analysis by TaqManeal-time PCR

Total DNA was extracted from the gills of dead crayfish, using annimal genomic DNA mini-prep kit (Sangon, Shanghai). The primeret VP28-FW and VP28-RV (5′-CGCACAGACAATATCGAGAC-3′/5′-TCTCAGTGCCAGAGTAGGT-3′), amplifying portion of the WSSVP28 gene, was used to screen for WSSV-positive animals. PCR waserformed with the VP28 primer set using the following protocol:

min at 94 ◦C followed by 35 cycles at 94 ◦C for 1 min, 55 ◦C for min and 72 ◦C for 1 min. The PCR products were analyzed by elec-rophoresis on 1% agarose gels stained with ethidium bromide andisualized by ultraviolet transillumination.

TaqMan real-time PCR was performed using a Perfect Realime premix (Takara, Japan) containing a high-performance Taqntibody, Takara Ex Taq HS, for hotstart real-time PCR. Primer 3oftware was used to design primers and the TaqMan probe withhe WSSV whole sequence (GenBank accession no. AF332093).

rimers WSSV-RT1 (5′-TTGGTTTCATGCCCGAGATT-3′) and WSSV-T2 (5′-CCTTGGTCAGCCCCTTGA-3′) produced a fragment of 154 bpfter amplification. The TaqMan probe was synthesized andabeled with the fluorescent dye 5-carboxyfluorescein (FAM)

ig. 2. Amplification curve showing 10 fold serial dilutions of the standard WSSV sampl × 106; (5) 1 × 105; (6) 1 × 104; (7) 1 × 103.

l Methods 184 (2012) 121– 124

(5′-FAM-TGCTGCCGTCTCCAA-TAMRA-3′). The reaction mixtureconsisted of a DNA aliquot of 200 nM of each primer, 100 nM of eachTaqMan probe, and 1× PCR buffer containing DNA polymerase ina final reaction volume of 20 �l. PCR amplification was performedfor 4 min at 50 ◦C, followed by 45 cycles of 45 s at 95 ◦C, 45 s at 52 ◦Cand 45 s at 72 ◦C. Thermal cycling was performed on an iCycle IQ5real-time PCR detection system (Bio-RAD, USA).

2.4. WSSV challenge and measurement of viral infectivity (LD50)

In this study, 30 crayfish per group were used for determina-tion of the infectivity (lethal dose 50%: LD50) of WSSV. To measurethe infectivity of this virus-containing fluid, 10-fold serial dilu-tions were made from 101 to 106 and filtered, using a Milliporefilter (pore size 0.45 mm). These diluted fluids were measured byTaqMan real-time PCR, after which they were administered byintramuscular (IM) injection, individually into 30 healthy crayfish,at a dose of 0.1 mL/crayfish. In the negative control group, crayfishwere IM injected with a TNE buffer at the same dose. The mortality,and clinical symptoms, of the crayfish were observed daily for thefollowing 21 days.

3. Results

3.1. Standard curve and DNA copies detection of WSSV titration

A TaqMan real-time PCR was used to quantify the WSSV copiesin the serial dilutions (101–106) of the original viral fluid. The stan-dard curve and detection limitation are shown in Fig. 1. Stronglinear correlations (R2 > 0.998) were obtained between the thresh-old cycles (Ct) and the target plasmid standard, ranging from1 × 103 to 1 × 109 WSSV copies in PCR, with a high reactionefficiency (E = 0.998) and proper slope (M = −3.326) (Fig. 2). Theamplification curves (Fig. 3) show results from seven dilutions ofstandard samples (Fig. 3). However, the negative control did not

show any amplification for each run. The 10-fold serial dilutions ofWSSV (from 101 to 106), using TaqMan real-time PCR, were thendetected for the DNA copies of the genomic DNA of crayfish. Thisresult indicates that WSSV copies were detected from 1.365 × 104

es. Numbers near lines (WSSV copies/�L): (1) 1 × 109; (2) 1 × 108; (3) 1 × 107; (4)

F. Zhu, H. Quan / Journal of Virological Methods 184 (2012) 121– 124 123

Fig. 3. The cumulative mortality of WSSV-infected crayfish injected with 101–106 dilutishowed the means of triplicate assays within ± 1% standard deviation.

Table 1The experimental WSSV infection in P. clarkii.

WSSV titration DNA copy(copies/mL)

Dead/tested Mortality (%)

101 dilution 1.129 × 109 30/30 100102 dilution 1.321 × 108 30/30 100103 dilution 1.214 × 107 30/30 100104 dilution 1.475 × 106 30/30 100

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10 dilution 1.524 × 10 18/30 60106 dilution 1.365 × 104 6/30 20Control – 0/30 0

o 1.129 × 109 copies/�l in the serial dilutions (101–106) of theriginal viral fluid.

.2. WSSV infection and measurement of viral infectivity (LD50)

The results from the viral infectivity (LD50) test, measured inerms of the mortality of infected crayfish, indicated 100% mortalityn the 101–104 dilution groups, and 60% mortality in the 105 dilu-ion group (Table 1 and Fig. 3). The LD50 value of the original viraluid was therefore estimated to be at a dilution of about 105. Thiseant that we could use a 104 dilution of WSSV as the challenge

ose in the challenge test that followed, which indicated that WSSVt such a dosage could result in 100% mortality of infected crayfishhen the DNA copies were above 1.475 × 106 copies/�l. Concen-

rations of 1.524 × 105 copies/�l WSSV would cause 60% mortalityt 21 days post challenge (dpc) (Table 1).

. Discussion

In previous studies, TaqMan real-time PCR was used to quan-ify WSSV infection in wild and farmed shrimp species, such as. chinensis, P. monodon, and L. vannamei (Jang et al., 2009; Fouzit al., 2010; Meng et al., 2010). In this investigation, which involvederial dilutions, WSSV copies were detected at concentrations from

× 103 to 1 × 109 copies/�l. In an optimal PCR mixture WSSV copiesould be detected with the genomic DNA in one reaction, indicating

large dynamic range and an assay of high sensitivity. This technol-gy provides a novel standard method of quantifying WSSV copies

or determining the correct challenge dose of WSSV in experimentalnfection tests.

The LD50 of WSSV is usually assessed by means of challengexperiments. Our tests, involving serial dilutions, can be used to

ons of WSSV. The groups used for injection were shown at the bottom. Each point

determine the levels at which 50% mortality occurs in shrimp orcrayfish. The highest dilution at which a mortality of 100% occurs,can also be used in challenge experiments (Prior et al., 2003; Zhuet al., 2009). The present results showed 100% mortality at a 10–104

dilution of WSSV and LD50 was estimated to be at a dilution of about105 (1.524 × 105 copies/�l), which showed 60% mortality (Table 1and Fig. 3). We therefore recommend the 104 dilution (1.475 × 106

copies/�l) of WSSV as the challenge dose in the following challengeexperiment of crayfish. Through the TaqMan real-time PCR, anyWSSV fluid which is above 1.475 × 106 copies/�l can be used as thechallenge dose for crayfish. The TaqMan real-time PCR can also beused to determine the correct challenge doses in other crustaceanspecies, such as shrimp.

This investigation showed that WSSV copies can be detectedat concentrations from 1 × 103 to 1 × 109 copies/�l using Taq-Man real-time PCR, indicating the large range of the assay. Thistechnology provides an efficient method for quantifying WSSV viadilutions. Compared with methods involving viral infectivity (LD50)measured using challenge experiments, more precise estimates ofthe lethal dose (as a challenge dose) were obtained by means ofTaqMan real-time PCR.

Acknowledgments

This work was financially supported by National Natural ScienceFoundation of China (31001127) and the Education DepartmentResearch Project of Zhejiang Province, China (No.Y200908288). Theauthors would like to thank Bin Zhi for his valuable contribution forthe work.

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