A new method for quantifying white spot syndrome virus: Experimental ...

Journal of Virological Methods 184 (2012) 121–124

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Short communication

A new method for quantifying white spot syndrome virus: Experimental challenge dose using TaqMan real-time PCR assay Fei Zhu a,∗ , Haizhi Quan b a b

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

a b s t r a c t Article history: Received 29 November 2011 Received in revised form 19 May 2012 Accepted 24 May 2012 Available online 1 June 2012 Keywords: TaqMan real-time PCR White spot syndrome virus Procambarus clarkii Challenge dose Experimental methods

White spot syndrome virus (WSSV) is an important pathogen in shrimp aquaculture. The susceptibility of 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 showed that WSSV copies could be detected at concentrations from 1.365 × 104 to 1.129 × 109 copies/␮l. The viral infectivity (LD50 ), measured as the mortality of infected crayfish, indicated 60% mortality in the 105 dilution group (1.524 × 105 copies/␮l). TaqMan real-time PCR represents a novel standard method, based on the by quantitation of WSSV copies, for determining the appropriate concentration of WSSV for use in infection experiments. © 2012 Elsevier B.V. All rights reserved.

1. Introduction White spot syndrome virus (WSSV), which was first discovered in Taiwan in 1992, has caused mass mortalities and devastating production losses to shrimp farming over many areas (Huang et al., 1995; Wang et al., 1995; Wongteerasupaya et al., 1995; Lightner, 1996). WSSV possesses a large DNA genome of about 300 kb, and genome analysis has shown that WSSV may be the sole member of the monotypic family Nimaviridae, genus Whispovirus (Van-Hulten et al., 2001; Yang et al., 2001; Vlak et al., 2005). WSSV is known to infect many crustacean species, including crayfish (Hossain et al., 2001; Lo et al., 1996). Baumgartner et al. (2009) recently found that both farmed and wild Procambarus clarkii in Louisiana (USA) were natural hosts for WSSV. Crayfish, such as Cherax quadricarinatus have also been used as experimental hosts for WSSV (Shi et al., 2000). A new real-time PCR technique, using TaqMan probe fluorescence, has been used recently to quantify the copy number of a particular target segment of nucleic acid, based on monitoring the increase in fluorescence. In previous studies, TaqMan real-time PCR was used to successfully quantify WSSV in various shrimp species, such as Penaeus monodon, Litopenaeus vannamei and Fenneropenaeus chinensis (Jang et al., 2009; Fouzi et al., 2010; Meng et al., 2010). In this investigation, a quantitative assay, involving a viral

∗ Corresponding author. Tel.: +86 571 63740815; fax: +86 571 63740815. E-mail address: [email protected] (F. Zhu). 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.05.026

titration of WSSV in P. clarkii, was described and determine precise lethal dosages for use in challenge experiments. 2. Materials and methods 2.1. Crayfish Crayfish (P. clarkii) of approximately 20 g were reared at 25 ± 1 ◦ C. They were kept in tanks with sand-filtered and ozonetreated water and fed with commercial pellet feed at 5% of body weight per day. The appendages from individuals selected at random were subjected to PCR assays to ensure that the crayfish were WSSV-free prior to the experimental challenge. 2.2. WSSV stock White spot syndrome virus-infected shrimp, F. chinensis, were collected from shrimp farms located near Ningbo, China. Ten grams of infected tissues (from the gills and the tail muscle) were homogenized in 500 mL TNE buffer (50 mM Tris–HCl, 400 mM NaCl, 5 mM EDTA, pH 7.5) containing a combination of protease inhibitors — (1 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM benzamidine, and 1 mM Na2 S2 O5 ) — 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 and filtrated using a Millipore filter (pore size 0.45 mm). The filtrate represented the original viral fluid used for further challenge tests.

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(5 -FAM-TGCTGCCGTCTCCAA-TAMRA-3 ). The reaction mixture consisted of a DNA aliquot of 200 nM of each primer, 100 nM of each TaqMan probe, and 1× PCR buffer containing DNA polymerase in a final reaction volume of 20 ␮l. PCR amplification was performed for 4 min at 50 ◦ C, followed by 45 cycles of 45 s at 95 ◦ C, 45 s at 52 ◦ C and 45 s at 72 ◦ C. Thermal cycling was performed on an iCycle IQ5 real-time PCR detection system (Bio-RAD, USA). 2.4. WSSV challenge and measurement of viral infectivity (LD50 )

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

2.3. WSSV detection by PCR and quantitative analysis by TaqMan real-time PCR Total DNA was extracted from the gills of dead crayfish, using an animal genomic DNA mini-prep kit (Sangon, Shanghai). The primer set VP28-FW and VP28-RV (5 -CGCACAGACAATATCGAGAC-3 /5 GTCTCAGTGCCAGAGTAGGT-3 ), amplifying portion of the WSSV VP28 gene, was used to screen for WSSV-positive animals. PCR was performed with the VP28 primer set using the following protocol: 5 min at 94 ◦ C followed by 35 cycles at 94 ◦ C for 1 min, 55 ◦ C for 1 min and 72 ◦ C for 1 min. The PCR products were analyzed by electrophoresis on 1% agarose gels stained with ethidium bromide and visualized by ultraviolet transillumination. TaqMan real-time PCR was performed using a Perfect Real Time premix (Takara, Japan) containing a high-performance Taq antibody, Takara Ex Taq HS, for hotstart real-time PCR. Primer 3 software was used to design primers and the TaqMan probe with the WSSV whole sequence (GenBank accession no. AF332093). Primers WSSV-RT1 (5 -TTGGTTTCATGCCCGAGATT-3 ) and WSSVRT2 (5 -CCTTGGTCAGCCCCTTGA-3 ) produced a fragment of 154 bp after amplification. The TaqMan probe was synthesized and labeled with the fluorescent dye 5-carboxyfluorescein (FAM)

In this study, 30 crayfish per group were used for determination of the infectivity (lethal dose 50%: LD50 ) of WSSV. To measure the infectivity of this virus-containing fluid, 10-fold serial dilutions were made from 101 to 106 and filtered, using a Millipore filter (pore size 0.45 mm). These diluted fluids were measured by TaqMan real-time PCR, after which they were administered by intramuscular (IM) injection, individually into 30 healthy crayfish, at a dose of 0.1 mL/crayfish. In the negative control group, crayfish were IM injected with a TNE buffer at the same dose. The mortality, and clinical symptoms, of the crayfish were observed daily for the following 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 copies in the serial dilutions (101 –106 ) of the original viral fluid. The standard curve and detection limitation are shown in Fig. 1. Strong linear correlations (R2 > 0.998) were obtained between the threshold cycles (Ct) and the target plasmid standard, ranging from 1 × 103 to 1 × 109 WSSV copies in PCR, with a high reaction efficiency (E = 0.998) and proper slope (M = −3.326) (Fig. 2). The amplification curves (Fig. 3) show results from seven dilutions of standard samples (Fig. 3). However, the negative control did not show any amplification for each run. The 10-fold serial dilutions of WSSV (from 101 to 106 ), using TaqMan real-time PCR, were then detected for the DNA copies of the genomic DNA of crayfish. This result indicates that WSSV copies were detected from 1.365 × 104

Fig. 2. Amplification curve showing 10 fold serial dilutions of the standard WSSV samples. Numbers near lines (WSSV copies/␮L): (1) 1 × 109 ; (2) 1 × 108 ; (3) 1 × 107 ; (4) 1 × 106 ; (5) 1 × 105 ; (6) 1 × 104 ; (7) 1 × 103 .


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Fig. 3. The cumulative mortality of WSSV-infected crayfish injected with 101 –106 dilutions of WSSV. The groups used for injection were shown at the bottom. Each point showed the means of triplicate assays within ± 1% standard deviation.

Table 1 The experimental WSSV infection in P. clarkii. WSSV titration

DNA copy (copies/mL)

Dead/tested

Mortality (%)

101 dilution 102 dilution 103 dilution 104 dilution 105 dilution 106 dilution Control

1.129 × 109 1.321 × 108 1.214 × 107 1.475 × 106 1.524 × 105 1.365 × 104 –

30/30 30/30 30/30 30/30 18/30 6/30 0/30

100 100 100 100 60 20 0

to 1.129 × 109 copies/␮l in the serial dilutions (101 –106 ) of the original viral fluid. 3.2. WSSV infection and measurement of viral infectivity (LD50 ) The results from the viral infectivity (LD50 ) test, measured in terms of the mortality of infected crayfish, indicated 100% mortality in the 101 –104 dilution groups, and 60% mortality in the 105 dilution group (Table 1 and Fig. 3). The LD50 value of the original viral fluid was therefore estimated to be at a dilution of about 105 . This meant that we could use a 104 dilution of WSSV as the challenge dose in the challenge test that followed, which indicated that WSSV at such a dosage could result in 100% mortality of infected crayfish when the DNA copies were above 1.475 × 106 copies/␮l. Concentrations of 1.524 × 105 copies/␮l WSSV would cause 60% mortality at 21 days post challenge (dpc) (Table 1). 4. Discussion In previous studies, TaqMan real-time PCR was used to quantify WSSV infection in wild and farmed shrimp species, such as F. chinensis, P. monodon, and L. vannamei (Jang et al., 2009; Fouzi et al., 2010; Meng et al., 2010). In this investigation, which involved serial dilutions, WSSV copies were detected at concentrations from 1 × 103 to 1 × 109 copies/␮l. In an optimal PCR mixture WSSV copies could be detected with the genomic DNA in one reaction, indicating a large dynamic range and an assay of high sensitivity. This technology provides a novel standard method of quantifying WSSV copies for determining the correct challenge dose of WSSV in experimental infection tests. The LD50 of WSSV is usually assessed by means of challenge experiments. Our tests, involving serial dilutions, can be used to

determine the levels at which 50% mortality occurs in shrimp or crayfish. The highest dilution at which a mortality of 100% occurs, can also be used in challenge experiments (Prior et al., 2003; Zhu et 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 about 105 (1.524 × 105 copies/␮l), which showed 60% mortality (Table 1 and Fig. 3). We therefore recommend the 104 dilution (1.475 × 106 copies/␮l) of WSSV as the challenge dose in the following challenge experiment of crayfish. Through the TaqMan real-time PCR, any WSSV fluid which is above 1.475 × 106 copies/␮l can be used as the challenge dose for crayfish. The TaqMan real-time PCR can also be used to determine the correct challenge doses in other crustacean species, such as shrimp. This investigation showed that WSSV copies can be detected at concentrations from 1 × 103 to 1 × 109 copies/␮l using TaqMan real-time PCR, indicating the large range of the assay. This technology provides an efficient method for quantifying WSSV via dilutions. Compared with methods involving viral infectivity (LD50 ) measured using challenge experiments, more precise estimates of the lethal dose (as a challenge dose) were obtained by means of TaqMan real-time PCR. Acknowledgments This work was financially supported by National Natural Science Foundation of China (31001127) and the Education Department Research Project of Zhejiang Province, China (No.Y200908288). The authors would like to thank Bin Zhi for his valuable contribution for the work. References Baumgartner, W.A., Hawke, J.P., Bowles, K., Varner, P.W., Hasson, K.W., 2009. Primary diagnosis and surveillance of white spot syndrome virus in wild and farmed crawfish (Procambarus clarkii, P. zonangulus) in Louisiana, USA. Disease of Aquatic Organisms 85, 15–22. Fouzi, M., Shariff, M., Omar, A.R., Yusoff, F.M., Tan, S.W., 2010. TaqMan real-time PCR assay for relative quantification of white spot syndrome virus infection in Penaeus monodon Fabricius exposed to ammonia. Journal of Fish Diseases 33, 931–938. Hossain, S., Chakraborty, A., Joseph, B., Otta, S.K., Karunasagar, I., Karunasagar, I., 2001. Detection of new hosts for white spot syndrome virus of shrimp using nested polymerase chain reaction. Aquaculture 198, 1–11. Huang, J., Cai, S., Song, X., Wang, C., Yu, J., Yang, C., 1995. Study on artificial infection for Penaeus chinensis by the pathogen of the explosive epidemic disease of shrimp. Marine Fisheries Research 16, 51–58.

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