doi:10.1111/jfd.12156
Journal of Fish Diseases 2014, 37, 703–710
Production of recombinant capsid protein of Macrobrachium rosenbergii nodavirus (r-MCP43) of giant freshwater prawn, M. rosenbergii (de Man) for immunological diagnostic methods M A Farook, N Madan, G Taju, S Abdul Majeed, K S N Nambi, N Sundar Raj, S Vimal and A S Sahul Hameed OIE Reference Laboratory for WTD, Department of Zoology, C. Abdul Hakeem College, Melvisharam, India
Abstract
White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a serious problem in prawn hatcheries. The gene for capsid protein of MrNV (MCP43) was cloned into pRSET B expression vector. The MCP43 protein was expressed as a protein with a 6-histidine tag in Escherichia coli GJ1158 with NaCl induction. This recombinant protein, which was used to raise the antiserum in rabbits, recognized capsid protein in different WTD-infected post-larvae and adult prawn. Various immunological methods such as Western blot, dot blot and ELISA techniques were employed to detect MrNV in infected samples using the antiserum raised against recombinant MCP43 of MrNV. The dot blot assay using anti-rMCP43 was found to be capable of detecting MrNV in WTD-infected post-larvae as early as at 24 h post-infection. The antiserum raised against r-MCP43 could detect the MrNV in the infected samples at the level of 100 pg of total protein. The capsid protein of MrNV estimated by ELISA using anti-rMCP43 and pure r-MCP43 as a standard was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. The results of immunological diagnostic methods employed in this study were compared with that of RT-PCR to test the efficiency of Correspondence A S Sahul Hameed, OIE Reference Laboratory for WTD, Department of Zoology, C. Abdul Hakeem College, Melvisharam 632509, Tamil Nadu, India. (e-mail: [email protected]) Ó 2013 John Wiley & Sons Ltd
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antiserum raised against r-MCP43 for the detection of MrNV. The Western blot, dot blot and ELISA detected all MrNV-positive coded samples as detected by RT-PCR. Keywords: Dot blot, ELISA, Macrobrachium rosenbergii nodavirus, polyclonal antiserum, recombinant capsid protein, Western blot.
Introduction
Macrobrachium rosenbergii is an important cultivable crustacean species, being cultured in different parts of the world including India. Disease caused by infectious agents is responsible for severe economic loss in aquaculture industry worldwide, and it is also true in the case of giant freshwater prawn culture. Generally, the freshwater prawn has been considered as hardy and resistant to infectious diseases. However, viral and bacterial diseases have been reported in different life stages of freshwater prawn (Brock 1988; Anderson et al. 1990; Arcier et al. 1999; Tung, Wang & Chen 1999; Sahul Hameed, Xavier Charles & Anilkumar 2000; Romestand & Bonami 2003; Sahul Hameed et al. 2004a; Hsieh et al. 2006; Wang et al. 2007). Among these diseases, a viral disease namely white tail disease caused by M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) is responsible for huge mortality in post-larval and juvenile stages, and its occurrence has been reported in different geographical regions such as French West Indies (Arcier et al. 1999), China
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(Qian et al. 2003), India (Sahul Hameed et al. 2004a), Thailand (Yoganandhan et al. 2006), Taiwan (Wang et al. 2008) and Australia (Owens et al. 2009). MrNV is small, icosahedral, nonenveloped particle with a diameter of 26–27 nm. Its genome consists of two pieces of ssRNA (RNA1 with size of 2.9 and RNA2 of 1.26 kb). This viral capsid contains a single polypeptide of 43 kDa. Extra small virus is a virus-like particle, icosahedral in shape and 15 nm in diameter, and its genome consists of a linear ssRNA of 0.9 kb encoding the structural proteins of 16 and 17 kDa (Qian et al. 2003). The diagnostic methods for detecting these viruses include RT-PCR (Sri Widada et al. 2003; Sahul Hameed et al. 2004a), loop-mediated isothermal amplification (LAMP) (Pillai, Bonami & Sri Widada 2006; Puthawibool et al. 2010), ELISA (Romestand & Bonami 2003; Qian et al. 2006; Sahul Hameed et al. 2011), in situ hybridization method (Sri Widada et al. 2003), dot blot hybridization (Sri Widada et al. 2004) and histopathology (Arcier et al. 1999). Sahul Hameed et al. (2011) have evaluated all nucleic acid and antibody-based diagnostics such as RT-PCR, nested RT-PCR, Western blot and ELISA for early detection of these viruses in experimentally infected post-larvae of freshwater prawn. Isolation and separation of MrNV and XSV is a laborious and time-consuming process. Moreover, pure viral particles still have contamination with host tissue after purification obtaining large quantity of pure viral particles for raising antiserum for developing diagnostics is also problematic. Recombinant DNA technology would be very useful to get the pure form of viral capsid protein. In addition will also be useful to get antiserum for developing immunological-based diagnostics for field application. In this context, an attempt was made in this study to clone the capsid gene of MrNV in prokaryotic vector (pRSET B) for production of recombinant capsid protein and subsequently raise antiserum for evaluating its efficiency to detect MrNV in infected samples.
Materials and methods
MrNV inoculum preparation The viral inoculum was prepared from infected post-larvae of prawn. The infected post-larvae with prominent sign of whitish muscle in the Ó 2013 John Wiley & Sons Ltd
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abdominal region were collected from hatcheries located near Nellore and Kakinada (Andhra Pradesh) and Bhubaneswar (Orissa). The postlarvae were washed thoroughly in sterile saline solution, transferred to sterile tubes for transport to the laboratory on dry ice and then stored at 80 °C. Frozen infected post-larvae were thawed and homogenized in a sterile homogenizer. A 10% (w/v) suspension was made with TN buffer (20 mM Tris-HCl and 0.4 M NaCl, pH 7.4). The homogenate was centrifuged at 4000 g for 20 min at 4 °C, and its supernatant was recentrifuged at 10 000 g for 20 min at 4 °C before the final supernatant was filtered through a 0.22 lm millipore membrane filter. The filtrate was then stored at 20 °C for infectivity studies and the isolation of capsid gene. The presence of MrNV and XSV in the samples was confirmed by RT-PCR. Extraction of total RNA, RT-PCR and cloning of MrNV capsid protein To extract the total RNA, 100 mg of whole postlarvae was homogenized in 1 mL of TN buffer (20 mM Tris-HCl, 0.4 M NaCl, pH 7.4). The homogenate was centrifuged at 12 000 g for 15 min at room temperature. The supernatant was collected and referred to as crude tissue extract. Total RNA was extracted using TRIzol reagent (Invitrogen). Briefly, 1 mL of TRIzol reagent was added to 200 lL of crude tissue extract and mixed thoroughly. After 5 min of incubation at room temperature, 0.2 mL of chloroform was added. The sample was vigorously shaken for 2–3 min at room temperature then centrifuged at 12 000 g for 15 min at room temperature. RNA was precipitated from the aqueous phase with isopropanol, washed with 70% ethanol and dissolved in 50 lL of sterile water. Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out using total RNA extracted from the sample to isolate capsid gene of MrNV. The RT-PCR analysis was carried out using the Reverse-ITTM 1-step RT-PCR kit (ABgene), allowing reverse transcription (RT) and amplification to be performed in a single reaction tube. Primers used to detect MrNV include published primers (Sahul Hameed et al. 2004a). The gene coding for MCP43 was amplified from Indian isolate of MrNV by RT-PCR with gene-specific primers designed based on the nucleotide sequence of MrNV available in GenBank (accession no. AY 222840). The sequence of the primers with the
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restriction sites and the corresponding annealing temperatures used to amplify the MCP43gene were 5-CGCGGATCCGATGGCT AGAGGTAAACA AA-3 (forward) and 5-CCG GAATTCCGCTAA TTATTGCCGACGA TA-3 (reverse) with BamHI and EcoRI restriction sites as indicated by bold italic letters. Reactions were performed in 50 lL RT-PCR buffer containing 20 pmol of each primer and RNA template, using the following steps: RT at 52 °C for 30 min; denaturation at 95 °C for 2 min followed by 30 cycles of denaturation at 95 °C for 40 s, annealing at 55 °C for 40 s and elongation at 72 °C for 1 min, ending with an additional elongation step of 10 min at 72 °C. Expression and purification of MCP gene in Escherichia coli The PCR fragment containing the MCP43 gene was cloned into pRSET B expression vector (Invitrogen) at BamHI and EcoRI sites and confirmed by BamHI and EcoRI digestion. The plasmid was transformed into E. coli GJ1158 (Bhandari & Gowrishankar 1997), and transformants were screened by PCR. MCP43 was expressed as a fusion protein with a 6-histidine tag in E. coli GJ1158 strain according to the procedure described by Bhandari & Gowrishankar (1997). Briefly, the culture was grown at 37 °C in lowosmolarity LB medium (Luria–Bertani medium with NaCl omitted, containing [L 1] 10 g of tryptone (casein enzyme hydrolysate) and 5 g of yeast extract [pH adjusted to 7.3 with NaOH]) supplemented with ampicillin. The cells were induced by addition of NaCl (from a 5 M stock) at a final concentration of 0.3 M. The expression of recombinant MCP43-histidine fusion protein was confirmed by Western blot using anti-rMCP43 antiserum and purified by affinity chromatography using a nickel–iminodiacetic acid (Ni-IDA) column according to the manufacturer’s instructions (Bio-Rad). Preparation of antibody Polyclonal antiserum against r-MCP43 was raised in white rabbits (2.5–3.0 kg) using purified protein emulsified with Freund’s complete adjuvant. The antiserum was collected from the immunized rabbits after completion of a standard immunization trial. Western blot was performed to determine the expression of r-MCP43 protein. ELISA and dot Ó 2013 John Wiley & Sons Ltd
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blot were carried out to determine the titres of antiserum against r-MCP43. Alkaline phosphatase–conjugated goat anti-rabbit IgG obtained from Sigma-Aldrich was used to detect r-MrNV and MrNV in WTD-infected samples. Antigen was replaced by PBS in negative control assays. Infectivity experiments The infectivity experiment was carried out by immersion challenge in post-larvae and intramuscular injection in adult prawn. In the immersion challenge, the post-larvae (50 Nos.) of freshwater prawn were placed in a 5-L beaker containing freshwater with continuous aeration. The beakers were covered to prevent contamination. The postlarvae were fed with Artemia nauplii. The inoculum prepared as mentioned previously was added to the water at a volume equal to 0.1% of the total rearing medium (1 mL L 1) (Venegas et al. 1999; Chen et al. 2000; Sahul Hameed et al. 2004a). Control groups were exposed to tissue filtrates (0.1%) prepared from healthy post-larvae of freshwater prawn collected from a freshwater prawn hatchery located near Chennai where there was no report of WTD. Samples were collected at different day intervals (1, 2, 3, 4, 5, 6, 7, 8 d p.i. and moribund stage). Twelve adult prawns were placed into four tanks and injected intramuscularly with inoculum prepared from WTD-infected PL of prawn. Different tissue samples were collected on 3 day post-injection for various analyses. Western blot Western blot analysis was carried out to test the efficiency of antiserum raised against r-MCP43 to detect MrNV in different WTD-infected samples which include post-larval samples collected at different time intervals of post-infection and different organs (gill tissue, muscle, heart tissue and pleopods) of adult prawn injected with MrNV. After separation on SDS-PAGE gel, the proteins were transferred onto a nitrocellulose membrane (Macherey-Nagel). After transfer, the membrane was blocked in blocking buffer at 4 °C overnight followed by incubation with primary antiserum (rabbit anti-r-MCP43 IgG) overnight. Subsequently, the membrane was incubated in ALP-conjugated goat anti-rabbit IgG (Sigma) for 1 h, and MrNV was detected with a substrate solution of 4-nitroblue tetrazolium
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chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Dot blot Samples of post-larvae exposed to MrNV at different time intervals were analysed to detect MrNV by dot blot assay using the antiserum raised against r-MCP43. The nitrocellulose membrane was cut to the desired size, soaked in PBS for 15 min and then mounted onto a dot blot apparatus. 5 lL of samples was blotted onto the membrane by applying a gentle vacuum. After transfer, the membrane was blocked in blocking buffer for 1 h at room temperature and incubated with primary antiserum (anti-r-MCP) for 1 h. Subsequently, the membrane was incubated in ALPconjugated goat anti-rabbit IgG (Sigma) for 1 h, and MrNV was detected with a substrate solution of 4-nitroblue tetrazolium chloride (NBT) and 5bromo-4-chloro-3-indolyl phosphate (BCIP) (SRL). Dot blot analysis was also used to determine the end point of titration for detection of MrNV in WTD-infected samples. After measuring the total protein, the infected sample was diluted 10-fold serial dilutions, and the diluted samples (diluted from 10 lg to 10 pg) were blotted onto the membrane. The blot was processed as mentioned above. ELISA An indirect ELISA was used in this study to detect MrNV in different infected samples using the polyclonal antibodies raised against r-MCP. The flat bottom ELISA plates were coated with different samples in coating buffer (0.1 M sodium carbonate, 0.25 M sodium bicarbonate, pH, 9.6) and kept overnight at 4 °C. Pure recombinant MCP43 protein (r-MCP43) of WTD was isolated by affinity chromatography using a nickel– iminodiacetic acid (Ni-IDA) column according to the manufacturer’s instructions (Bio-Rad) and used as a standard to quantify the viral protein in the samples. After coating, the plates were washed once with PBS and blocked with 2% BSA in PBS for 1 h at 37 °C. The plates were incubated with 100 lL of primary antibodies (final dilution 1:20 000) raised against r-MCP43 for 3 h at 37 °C. Then, the plates were washed with PBS-T (0.05% Tween 20) and PBS three times for 2 min each time and incubated with 100 lL of Ó 2013 John Wiley & Sons Ltd
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anti-rabbit IgG alkaline phosphatase conjugate (final dilution 1:20 000) for 1 h. The plates were washed with PBS-T and PBS three times for 2 min each time and developed with the substrate p-nitro phenyl phosphate in substrate buffer. The optical density was measured at 405 nm using an ELISA reader (Multiskan EX Microplate Photometer; Thermo Electron Corporation). Results
The gene encoding capsid protein of MrNV was cloned in prokaryotic expression vector, pRSET B, and the clone with MCP43 gene was designated as pRMCP43 (pRSET B-M. rosenbergii nodavirus capsid protein 43 kDa). The expression of r-MCP43 was confirmed by Western blot using monoclonal antibodies raised against histidine and polyclonal antibodies raised whole MrNV (Sahul Hameed et al. 2011). The purified r-MCP43 was used to raise the antiserum in rabbit, and the efficiency of this antiserum to detect MrNV in the samples was tested by Western blot. The results revealed that the anti-rMCP43 detected MrNV in experimentally and naturally WTD-infected postlarval samples by the appearance of a distinct band of 43 kDa in Western blot (Fig. 1). The antiserum raised against r-MCP43 successfully detected MrNV in different organs such as gill tissue, pleopods, muscle and heart tissue of experimentally WTD-infected prawn by Western blot (Fig. 2). The dot blot analysis carried out using the antiserum raised against r-MCP43 was found to be capable of detecting the MrNV in WTD-infected post-larvae at 24 h post-infection (Fig. 3). The intensity of band was found to be increased during the course of infection in the post-larvae. The intensity of band was high in the samples of 5 and 7 d p.i. and moribund (Fig. 3). The dot blot analysis using the anti-rMCP43 detected MrNV in WTD-infected samples at the level of 100 pg of total protein (Fig. 4). Beyond this level, the antiserum failed to detect the MrNV in the infected samples (Fig. 4). Indirect ELISA technique was applied to quantify the 43 kDa capsid protein of MrNV in WTD-infected post-larval samples collected at different time intervals of post-infection and also in different organs of MrNV-injected adult prawn using antiserum raised against r-MCP43 which was used as a standard protein. The 43 kDa capsid protein was estimated to be about 0.57 lg per
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Figure 3 Dot blot analysis for the detection of MrNV in WTD-infected post-larvae at different time intervals (day postinfection [d p.i.]) in time course infectivity experiment. P – Positive control; N – Negative control; 1 – 1st d.p.i.; 2 – 2nd d.p.i.; 3 – 3rd d.p.i.; 4 – 5th d.p.i.; 5 – 7th d.p.i.; and 6 – Moribund. Figure 1 Western blot analysis of pRMCP43 protein of MrNV. Lane M – 100 kDa protein marker; lane 1 – Uninfected PL of Macrobrachium rosenbergii (negative control); lane 2 – Experimentally WTD-infected PL of prawn (positive control); lane 3 – Purified r-MCP43 protein; lane 4 – Induced pRMCP43-GJ1158; lane 5 – Non-induced pRMCP43GJ1158; and lane 6 – WTD-infected PL sample of M. rosenbergii (wild sample).
Figure 2 Detection of WTD-MCP43 protein by Western blot using antiserum of r-MCP43 in different organs of WTD-infected adult prawn on 3 d.p.i. Lane M – 100 kDa protein marker; lane 1 – Negative control; lane 2 – Gill tissue; lane 3 – Muscle; lane 4 – Pleopods; and lane 5 – Heart.
0.1 mg of total protein in post-larvae exposed to MrNV after 24 h p.i., and this value increased gradually during the course of infection and reached the maximum level of 1.4 lg per 0.1 mg of total protein at moribund stage (Fig. 5). The 43 kDa capsid protein of MrNV was quantified about 0.9, 1.0, 0.8 and 0.85 lg per 0.1 mg of total protein, respectively, in gill, heart, muscle and pleopods of experimentally MrNV-injected adult giant freshwater prawn. The known MrNV-positive and negative samples were coded, and the coded samples were screened for MrNV using anti-rMCP43 by Western blot, dot blot and ELISA techniques. These results were compared with that of RT-PCR to test the efficiency of antiserum raised against r-MCP43 for the detection of MrNV. The Western blot, dot blot Ó 2013 John Wiley & Sons Ltd
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Figure 4 Dot blot analysis for end-point titration to detect MrNV in WTD-infected sample. P – Positive control; N – Negative control; 1 – 10 lg of total protein; 2 – 1 lg; 3 – 100 ng; 4 – 10 ng; 5 – 1 ng; 6 – 100 pg; 7 – 10 pg.
Figure 5 Quantification of MrNV protein of WTD in PL of Macrobrachium rosenbergii at different day intervals using purified r-MCP of WTD protein as standard. PC – Positive control, NC – Negative control, 1 – 1st d.p.i.; 2 – 2nd d.p.i.; 3 – 3rd d.p.i.; 4 – 4th d.p.i., 5 – 5th d.p.i.; 6 – 6th d.p.i.; 7 – 7th d.p.i.; 8 – 8th d.p.i.; and M – Moribund.
and ELISA detected all MrNV-positive coded samples as detected by RT-PCR (Table 1). Discussion
White tail disease caused by MrNV is responsible for huge economic loss in all prawn growing countries including India. The prawn production in India has been reduced to less than 2000 tonnes from 40 000 tonnes in 2003 due to various factors including WTD. The MrNV affects early life stages such as larvae, post-larvae and juveniles, and previous reports also revealed the possibility of vertical transmission of this virus from brooder to eggs (Sahul Hameed et al. 2004b; Sudhakaran et al. 2007). Screening of brooders for MrNV and
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Journal of Fish Diseases 2014, 37, 703–710
Table 1 Detection of MrNV in different known positive and negative coded samples using RT-PCR, Western blot, immunodot blot and ELISA assays Samples Negative control Positive control S. No.: 1 S. No.: 2 S. No.: 3 S. No.: 4 S. No.: 5 S. No.: 6 S. No.: 7 S. No.: 8 S. No.: 9 S. No.: 10
RT -PCR
ELISA
Immunodot blot
Western blot
Remarks
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XSV is essential to get disease free larvae and postlarvae. Commercial screening of the brooders by RT-PCR is costly and laborious. A simple, cost effective and moderate by sensitive diagnostic method for preliminary screening of prawn brooders would be very useful for prawn culture industry. Immunological-based diagnostic methods using the antiserum raised against whole MrNV was found to be very useful for preliminary screening. It was also found to be as sensitive as RT-PCR. Antiserum raised against purified MrNV has several limitations as observed by Romestand & Bonami (2003), Qian et al. (2006) & Sahul Hameed et al. (2011). Among these are the high cost and effort required for virus propagation which requires live animals due to lack of susceptible cell lines for this virus. White tail disease is caused by a combination of two viruses, MrNV and XSV, which have to be separated and purified for raising antiserum. Separation and purification process does not completely remove trace quantities of prawn tissue proteins that may be immunogenic. Use of recombinant DNA technology for the production of recombinant viral proteins overcomes these problems, and its purification is very simple using the histidine-tag purification system. In the present study, a bacterial host namely E. coli GJ1158 developed by Bhandari & Gowrishankar (1997) was used to produce recombinant capsid protein of MrNV (r-MCP43) with NaCl induction. This host system is very useful for the expression of cloned gene products, and the expression is under the control of osmoresponsive proU promoters. It is suitable for a variety of Ó 2013 John Wiley & Sons Ltd
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pET vectors that are used in BL21 (DE3) E. coli strains. The antiserum raised against recombinant MCP43 of MrNV was evaluated for its efficiency to detect MrNV in naturally and experimentally WTD-infected samples by different immunological methods such as Western blot, dot blot and ELISA. The anti-rMCP43 detected MrNV in different samples by Western blot as detected by the antiserum raised against MrNV (Romestand & Bonami 2003; Sahul Hameed et al. 2011). A similar study has been carried out by Yoganandhan et al. (2004) who studied the efficiency of antiserum raised against recombinant VP28 protein of white spot syndrome virus (WSSV) of shrimp to detect the WSSV in different infected samples. The anti-rMCP43 detected MrNV in gill, muscle, heart and pleopods of adult prawn, as well as post-larvae. This result revealed that all these organs including post-larvae are good sources for detection of MrNV using immunological diagnostics as observed by Sahul Hameed et al. (1998) & Yoganandhan et al. (2004) for WSSV. The dot blot assay using anti-rMCP43 was found to be capable of detecting MrNV in WTD-infected post-larvae as early as at 24 h post-infection (p.i.) as observed by Sahul Hameed et al. (2011) by Western blot and ELISA using the antiserum raised against MrNV. Similarly, Yoganandhan et al. (2004) used the antiserum raised against recombinant VP28 protein to detect WSSV in different organs of WSSV-infected shrimp by Western blot, and their results revealed the capability of this antiserum to detect WSSV within 24 h p.i. Nadala & Loh (1998) reported the detection of WSSV in experimentally infected white leg shrimp, Penaeus vannamei (Boone) at 41 h p.i. using the antiserum raised against WSSV. The antiserum raised against r-MCP43 detected MrNV in 100 pg of total protein prepared from WTD post-larvae by dot blot assay, whereas the anti-rVP28 detected WSSV in 5 ng of total haemolymph protein (Yoganandhan et al. 2004). The antiserum raised against whole WSSV detected WSSV in 800 ng of total haemolymph protein of WSSV-infected shrimp (Sahul Hameed et al. 1998). These studies clearly indicate the high efficiency of antiserum raised against recombinant viral protein for the detection of viral pathogens in comparison with antiserum raised against whole virus. In addition, production of antiserum against whole virus was proved to be difficult because of damaging of virus during
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purification, cross-reaction with host proteins and laborious for virus propagation (Nadala & Loh 2000; Chaivisuthangkura et al. 2004; Yoganandhan et al. 2004). The ELISA technique using the anti-rMCP43 was employed to quantify the capsid protein of MrNV in WTD-infected post-larvae at different day intervals after post-infection. The pure r-MCP43 was used as a standard to quantify the capsid protein of MrNV. The capsid protein was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. This indicates that the severity of WTD infection in post-larvae could be easily determined by ELISA using anti-rMCP43 and pure r-MCP43 as standard. The ELISA technique with pure r-MCP43 helped to determine the concentration of capsid protein of MrNV in different organs of MrNVinjected prawn, and the results revealed the high concentration of capsid protein in gill tissue, heart, pleopods and muscle. In the present study, we used known MrNVpositive and negative coded samples to compare our immunological diagnostic methods with RT-PCR detection system (Table 1). Results obtained were identical among Western blot, dot blot, ELISA and RT-PCR assays. These results indicate the possibility of using immunodot assay for field detection of MrNV in brooders, post-larvae and grow-out prawn which would be useful in the prawn culture industry. This kit would be economical, sensitive (equivalent to RT-PCR), rapid, very simple to perform and suitable for the development of a commercial detection kit at least for preliminary screening.
Bhandari P. & Gowrishankar J. (1997) An Escherichia coli host strain useful for efficient overproduction of cloned gene products with NaCl as the inducer. Journal of Bacteriology 179, 4403–4406. Brock J.A. (1988) Diseases and husbandry problems of cultured Macrobrachium rosenbergii. In: Disease Diagnosis and Control in North American Marine Aquaculture (ed. by C.J. Sindermann & D.U. Lightner), pp. 134–180. Elsevier, Amsterdam. Chaivisuthangkura P., Tangkhabuanbutra J., Longyant S., Sithigorngul W., Rukpratanporn S., Menasveta P. & Sithigorngul P. (2004) Monoclonal antibodies against a truncated viral envelope protein (VP28) can detect white spot syndrome virus (WSSV) infections in shrimp. Science Asia 30, 359–363. Chen L.L., Lo C.F., Chiu Y.L., Chang C.F. & Kou G.H. (2000) Natural and experimental infection of white spot syndrome virus (WSSV) in benthic larvae of mud crab Scylla serrata. Disease of Aquatic Organisms 40, 157–161. Hsieh C.Y., Chuang P.C., Chen L.C., Tu C., Chien M.S., Huang K.C., Kao H.F., Tung M.C. & Tsai S.S. (2006) Infectious hypodermal and hematopoietic necrosis virus (IHHNV) infections in giant freshwater prawn, Macrobrachium rosenbergii. Aquaculture 258, 73–79. Nadala .E.C.B. Jr & Loh P.C. (1998) A comparative study of three different isolates of white spot virus. Disease of Aquatic Organisms 33, 231–234. Nadala E.C.B. & Loh P.C. (2000) Dot-blot nitrocellulose enzyme immunoassays for the detection of white-spot virus and yellow-head virus of penaeid shrimp. Journal of Virological Methods 84, 175–179. Owens L., Fauce1 K.L., Juntunen K., Hayakijkosol O. & Zeng C. (2009) Macrobrachium rosenbergii nodavirus disease (white tail disease) in Australia. Disease of Aquatic Organisms 85, 175–180.
Acknowledgements
Pillai D., Bonami J.R. & Sri Widada J. (2006) Rapid detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), the pathogenic agents of white tail disease of Macrobrachium rosenbergii (De Man), by loop-mediated isothermal amplification. Journal of Fish Diseases 29, 275–283.
The authors are grateful to the Management of C. Abdul Hakeem College, Melvisharam, India, for providing the facilities to carry out this work. This work was funded by the Department of Biotechnology, Government of India, New Delhi, India.
Puthawibool T., Senapin S., Flegel T.W. & Kiatpathomchai W. (2010) Rapid and sensitive detection of Macrobrachium rosenbergii nodavirus in giant freshwater prawns by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick. Molecular and Cellular Probes 24, 244–249.
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Romestand B. & Bonami J.R. (2003) A sandwich enzyme linked immune sorbent assay (S-ELISA) for detection of MrNV in the giant freshwater prawn, Macrobrachium rosenbergii (de Man). Journal of Fish Diseases 26, 71–75. Sahul Hameed A.S., Anilkumar M., Stephen Raj M.L. & Jayaraman K. (1998) Studies on the pathogencity of systemic ectodermal and mesodermal baculovirus and its detection in shrimp by immunological methods. Aquaculture 160, 31–45. Sahul Hameed A.S., Xavier Charles M. & Anilkumar M. (2000) Tolerance of Macrobrachium rosenbergii to white spot syndrome virus. Aquaculture 183, 207–213. Sahul Hameed A.S., Yoganandhan K., Sri Widada J. & Bonami J.R. (2004a) Studies on the occurrence of Macrobrachium rosenbergii nodavirus and extra small viruslike particles associated with white tail disease of M. rosenbergii in India by RT-PCR detection. Aquaculture 238, 127–133. Sahul Hameed A.S., Yoganandhan K., Sri Widada J. & Bonami J.R. (2004b) Experimental transmission and tissue tropism of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus like-particles in Macrobrachium rosenbergii. Disease of Aquatic Organisms 62, 191–196. Sahul Hameed A.S., Ravi M., Farook M.A., Taju G., Hernandez-Herrera R.I. & Bonami J.R. (2011) Screening the post-larvae of Macrobrachium rosenbergii for early detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) by RT–PCR and immunological techniques. Aquaculture 317, 42–47. Sri Widada J., Durand S., Cambournac I., Qian D., Shi Z., Dejonghe E., Richard V. & Bonami J.R. (2003) Genomebased detection methods of Macrobrachium rosenbergii nodavirus, a pathogen of the giant freshwater prawn, Macrobrachium rosenbergii dot blot, in situ hybridization and RT-PCR. Journal of Fish Diseases 26, 583–590. Sri Widada J., Richard V., Shi Z., Qian D. & Bonami J.R. (2004) Dot-blot hybridization and RT-PCR detection of extra small virus (XSV) associated with white tail disease of prawn Macrobrachium rosenbergii. Disease of Aquatic Organisms 58, 83–87.
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Sudhakaran R., Ishaq Ahmed V.P., Haribabu P., Mukherjee S.C., Sri Widada J., Bonami J.R. & Sahul Hameed A.S. (2007) Experimental vertical transmission of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) from brooders to progeny in Macrobrachium rosenbergii and Artemia. Journal of Fish Diseases 30, 27–35. Tung C.W., Wang C.S. & Chen S.N. (1999) Histological and electron microscopic study on Macrobrachium muscle virus (MMV) infection in the giant freshwater prawn, Macrobrachium rosenbergii (de Man), cultured in Taiwan. Journal of Fish Diseases 22, 319–324. Venegas C.A., Nonaka L., Mushiake K., Nishizawa T. & Muroga K. (1999) Pathogenicity of penaeid rod-shaped DNA virus PRDV to kuruma prawn at different development stages. Fish Pathology 170, 179–194. Wang C.S., Chang J.S., Shih H.H. & Chen S.N. (2007) RT-PCR amplification and sequence analysis of Macrobrachium rosenbergii nodavirus and extra small virus (XSV) associated with white tail disease of M. rosenbergii (de Man) cultured in Taiwan. Journal of Fish Diseases 30, 127–132. Wang C.S., Chang J.S., Wen C.M., Shih H.H. & Chen S.N. (2008) Macrobrachium rosenbergii noda virus infection in M. rosenbergii (de Man) with white tail disease cultured in Taiwan. Journal of Fish Diseases 31, 415–422. Yoganandhan K., Syed Musthaq S., Narayanan R.B. & Sahul Hameed A.S. (2004) Production of polyclonal antiserum against recombinant VP28 protein and its application for the white spot syndrome virus in crustaceans. Journal of Fish Diseases 27, 517–522. Yoganandhan K., Leartvibhan M., Sriwongpuk S. & Limsuwan C. (2006) White tail disease of the giant freshwater prawn Macrobrachium rosenbergii in Thailand. Disease of Aquatic Organisms 69, 255–258. Received: 13 April 2013 Revision received: 10 June 2013 Accepted: 19 June 2013
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Production of recombinant capsid protein of Macrobrachium rosenbergii nodavirus (r-MCP43) of giant freshwater prawn, M. rosenbergii (de Man) for immunological diagnostic methods M A Farook, N Madan, G Taju, S Abdul Majeed, K S N Nambi, N Sundar Raj, S Vimal and A S Sahul Hameed OIE Reference Laboratory for WTD, Department of Zoology, C. Abdul Hakeem College, Melvisharam, India
Abstract
White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a serious problem in prawn hatcheries. The gene for capsid protein of MrNV (MCP43) was cloned into pRSET B expression vector. The MCP43 protein was expressed as a protein with a 6-histidine tag in Escherichia coli GJ1158 with NaCl induction. This recombinant protein, which was used to raise the antiserum in rabbits, recognized capsid protein in different WTD-infected post-larvae and adult prawn. Various immunological methods such as Western blot, dot blot and ELISA techniques were employed to detect MrNV in infected samples using the antiserum raised against recombinant MCP43 of MrNV. The dot blot assay using anti-rMCP43 was found to be capable of detecting MrNV in WTD-infected post-larvae as early as at 24 h post-infection. The antiserum raised against r-MCP43 could detect the MrNV in the infected samples at the level of 100 pg of total protein. The capsid protein of MrNV estimated by ELISA using anti-rMCP43 and pure r-MCP43 as a standard was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. The results of immunological diagnostic methods employed in this study were compared with that of RT-PCR to test the efficiency of Correspondence A S Sahul Hameed, OIE Reference Laboratory for WTD, Department of Zoology, C. Abdul Hakeem College, Melvisharam 632509, Tamil Nadu, India. (e-mail: [email protected]) Ó 2013 John Wiley & Sons Ltd
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antiserum raised against r-MCP43 for the detection of MrNV. The Western blot, dot blot and ELISA detected all MrNV-positive coded samples as detected by RT-PCR. Keywords: Dot blot, ELISA, Macrobrachium rosenbergii nodavirus, polyclonal antiserum, recombinant capsid protein, Western blot.
Introduction
Macrobrachium rosenbergii is an important cultivable crustacean species, being cultured in different parts of the world including India. Disease caused by infectious agents is responsible for severe economic loss in aquaculture industry worldwide, and it is also true in the case of giant freshwater prawn culture. Generally, the freshwater prawn has been considered as hardy and resistant to infectious diseases. However, viral and bacterial diseases have been reported in different life stages of freshwater prawn (Brock 1988; Anderson et al. 1990; Arcier et al. 1999; Tung, Wang & Chen 1999; Sahul Hameed, Xavier Charles & Anilkumar 2000; Romestand & Bonami 2003; Sahul Hameed et al. 2004a; Hsieh et al. 2006; Wang et al. 2007). Among these diseases, a viral disease namely white tail disease caused by M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) is responsible for huge mortality in post-larval and juvenile stages, and its occurrence has been reported in different geographical regions such as French West Indies (Arcier et al. 1999), China
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(Qian et al. 2003), India (Sahul Hameed et al. 2004a), Thailand (Yoganandhan et al. 2006), Taiwan (Wang et al. 2008) and Australia (Owens et al. 2009). MrNV is small, icosahedral, nonenveloped particle with a diameter of 26–27 nm. Its genome consists of two pieces of ssRNA (RNA1 with size of 2.9 and RNA2 of 1.26 kb). This viral capsid contains a single polypeptide of 43 kDa. Extra small virus is a virus-like particle, icosahedral in shape and 15 nm in diameter, and its genome consists of a linear ssRNA of 0.9 kb encoding the structural proteins of 16 and 17 kDa (Qian et al. 2003). The diagnostic methods for detecting these viruses include RT-PCR (Sri Widada et al. 2003; Sahul Hameed et al. 2004a), loop-mediated isothermal amplification (LAMP) (Pillai, Bonami & Sri Widada 2006; Puthawibool et al. 2010), ELISA (Romestand & Bonami 2003; Qian et al. 2006; Sahul Hameed et al. 2011), in situ hybridization method (Sri Widada et al. 2003), dot blot hybridization (Sri Widada et al. 2004) and histopathology (Arcier et al. 1999). Sahul Hameed et al. (2011) have evaluated all nucleic acid and antibody-based diagnostics such as RT-PCR, nested RT-PCR, Western blot and ELISA for early detection of these viruses in experimentally infected post-larvae of freshwater prawn. Isolation and separation of MrNV and XSV is a laborious and time-consuming process. Moreover, pure viral particles still have contamination with host tissue after purification obtaining large quantity of pure viral particles for raising antiserum for developing diagnostics is also problematic. Recombinant DNA technology would be very useful to get the pure form of viral capsid protein. In addition will also be useful to get antiserum for developing immunological-based diagnostics for field application. In this context, an attempt was made in this study to clone the capsid gene of MrNV in prokaryotic vector (pRSET B) for production of recombinant capsid protein and subsequently raise antiserum for evaluating its efficiency to detect MrNV in infected samples.
Materials and methods
MrNV inoculum preparation The viral inoculum was prepared from infected post-larvae of prawn. The infected post-larvae with prominent sign of whitish muscle in the Ó 2013 John Wiley & Sons Ltd
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abdominal region were collected from hatcheries located near Nellore and Kakinada (Andhra Pradesh) and Bhubaneswar (Orissa). The postlarvae were washed thoroughly in sterile saline solution, transferred to sterile tubes for transport to the laboratory on dry ice and then stored at 80 °C. Frozen infected post-larvae were thawed and homogenized in a sterile homogenizer. A 10% (w/v) suspension was made with TN buffer (20 mM Tris-HCl and 0.4 M NaCl, pH 7.4). The homogenate was centrifuged at 4000 g for 20 min at 4 °C, and its supernatant was recentrifuged at 10 000 g for 20 min at 4 °C before the final supernatant was filtered through a 0.22 lm millipore membrane filter. The filtrate was then stored at 20 °C for infectivity studies and the isolation of capsid gene. The presence of MrNV and XSV in the samples was confirmed by RT-PCR. Extraction of total RNA, RT-PCR and cloning of MrNV capsid protein To extract the total RNA, 100 mg of whole postlarvae was homogenized in 1 mL of TN buffer (20 mM Tris-HCl, 0.4 M NaCl, pH 7.4). The homogenate was centrifuged at 12 000 g for 15 min at room temperature. The supernatant was collected and referred to as crude tissue extract. Total RNA was extracted using TRIzol reagent (Invitrogen). Briefly, 1 mL of TRIzol reagent was added to 200 lL of crude tissue extract and mixed thoroughly. After 5 min of incubation at room temperature, 0.2 mL of chloroform was added. The sample was vigorously shaken for 2–3 min at room temperature then centrifuged at 12 000 g for 15 min at room temperature. RNA was precipitated from the aqueous phase with isopropanol, washed with 70% ethanol and dissolved in 50 lL of sterile water. Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out using total RNA extracted from the sample to isolate capsid gene of MrNV. The RT-PCR analysis was carried out using the Reverse-ITTM 1-step RT-PCR kit (ABgene), allowing reverse transcription (RT) and amplification to be performed in a single reaction tube. Primers used to detect MrNV include published primers (Sahul Hameed et al. 2004a). The gene coding for MCP43 was amplified from Indian isolate of MrNV by RT-PCR with gene-specific primers designed based on the nucleotide sequence of MrNV available in GenBank (accession no. AY 222840). The sequence of the primers with the
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restriction sites and the corresponding annealing temperatures used to amplify the MCP43gene were 5-CGCGGATCCGATGGCT AGAGGTAAACA AA-3 (forward) and 5-CCG GAATTCCGCTAA TTATTGCCGACGA TA-3 (reverse) with BamHI and EcoRI restriction sites as indicated by bold italic letters. Reactions were performed in 50 lL RT-PCR buffer containing 20 pmol of each primer and RNA template, using the following steps: RT at 52 °C for 30 min; denaturation at 95 °C for 2 min followed by 30 cycles of denaturation at 95 °C for 40 s, annealing at 55 °C for 40 s and elongation at 72 °C for 1 min, ending with an additional elongation step of 10 min at 72 °C. Expression and purification of MCP gene in Escherichia coli The PCR fragment containing the MCP43 gene was cloned into pRSET B expression vector (Invitrogen) at BamHI and EcoRI sites and confirmed by BamHI and EcoRI digestion. The plasmid was transformed into E. coli GJ1158 (Bhandari & Gowrishankar 1997), and transformants were screened by PCR. MCP43 was expressed as a fusion protein with a 6-histidine tag in E. coli GJ1158 strain according to the procedure described by Bhandari & Gowrishankar (1997). Briefly, the culture was grown at 37 °C in lowosmolarity LB medium (Luria–Bertani medium with NaCl omitted, containing [L 1] 10 g of tryptone (casein enzyme hydrolysate) and 5 g of yeast extract [pH adjusted to 7.3 with NaOH]) supplemented with ampicillin. The cells were induced by addition of NaCl (from a 5 M stock) at a final concentration of 0.3 M. The expression of recombinant MCP43-histidine fusion protein was confirmed by Western blot using anti-rMCP43 antiserum and purified by affinity chromatography using a nickel–iminodiacetic acid (Ni-IDA) column according to the manufacturer’s instructions (Bio-Rad). Preparation of antibody Polyclonal antiserum against r-MCP43 was raised in white rabbits (2.5–3.0 kg) using purified protein emulsified with Freund’s complete adjuvant. The antiserum was collected from the immunized rabbits after completion of a standard immunization trial. Western blot was performed to determine the expression of r-MCP43 protein. ELISA and dot Ó 2013 John Wiley & Sons Ltd
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blot were carried out to determine the titres of antiserum against r-MCP43. Alkaline phosphatase–conjugated goat anti-rabbit IgG obtained from Sigma-Aldrich was used to detect r-MrNV and MrNV in WTD-infected samples. Antigen was replaced by PBS in negative control assays. Infectivity experiments The infectivity experiment was carried out by immersion challenge in post-larvae and intramuscular injection in adult prawn. In the immersion challenge, the post-larvae (50 Nos.) of freshwater prawn were placed in a 5-L beaker containing freshwater with continuous aeration. The beakers were covered to prevent contamination. The postlarvae were fed with Artemia nauplii. The inoculum prepared as mentioned previously was added to the water at a volume equal to 0.1% of the total rearing medium (1 mL L 1) (Venegas et al. 1999; Chen et al. 2000; Sahul Hameed et al. 2004a). Control groups were exposed to tissue filtrates (0.1%) prepared from healthy post-larvae of freshwater prawn collected from a freshwater prawn hatchery located near Chennai where there was no report of WTD. Samples were collected at different day intervals (1, 2, 3, 4, 5, 6, 7, 8 d p.i. and moribund stage). Twelve adult prawns were placed into four tanks and injected intramuscularly with inoculum prepared from WTD-infected PL of prawn. Different tissue samples were collected on 3 day post-injection for various analyses. Western blot Western blot analysis was carried out to test the efficiency of antiserum raised against r-MCP43 to detect MrNV in different WTD-infected samples which include post-larval samples collected at different time intervals of post-infection and different organs (gill tissue, muscle, heart tissue and pleopods) of adult prawn injected with MrNV. After separation on SDS-PAGE gel, the proteins were transferred onto a nitrocellulose membrane (Macherey-Nagel). After transfer, the membrane was blocked in blocking buffer at 4 °C overnight followed by incubation with primary antiserum (rabbit anti-r-MCP43 IgG) overnight. Subsequently, the membrane was incubated in ALP-conjugated goat anti-rabbit IgG (Sigma) for 1 h, and MrNV was detected with a substrate solution of 4-nitroblue tetrazolium
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chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Dot blot Samples of post-larvae exposed to MrNV at different time intervals were analysed to detect MrNV by dot blot assay using the antiserum raised against r-MCP43. The nitrocellulose membrane was cut to the desired size, soaked in PBS for 15 min and then mounted onto a dot blot apparatus. 5 lL of samples was blotted onto the membrane by applying a gentle vacuum. After transfer, the membrane was blocked in blocking buffer for 1 h at room temperature and incubated with primary antiserum (anti-r-MCP) for 1 h. Subsequently, the membrane was incubated in ALPconjugated goat anti-rabbit IgG (Sigma) for 1 h, and MrNV was detected with a substrate solution of 4-nitroblue tetrazolium chloride (NBT) and 5bromo-4-chloro-3-indolyl phosphate (BCIP) (SRL). Dot blot analysis was also used to determine the end point of titration for detection of MrNV in WTD-infected samples. After measuring the total protein, the infected sample was diluted 10-fold serial dilutions, and the diluted samples (diluted from 10 lg to 10 pg) were blotted onto the membrane. The blot was processed as mentioned above. ELISA An indirect ELISA was used in this study to detect MrNV in different infected samples using the polyclonal antibodies raised against r-MCP. The flat bottom ELISA plates were coated with different samples in coating buffer (0.1 M sodium carbonate, 0.25 M sodium bicarbonate, pH, 9.6) and kept overnight at 4 °C. Pure recombinant MCP43 protein (r-MCP43) of WTD was isolated by affinity chromatography using a nickel– iminodiacetic acid (Ni-IDA) column according to the manufacturer’s instructions (Bio-Rad) and used as a standard to quantify the viral protein in the samples. After coating, the plates were washed once with PBS and blocked with 2% BSA in PBS for 1 h at 37 °C. The plates were incubated with 100 lL of primary antibodies (final dilution 1:20 000) raised against r-MCP43 for 3 h at 37 °C. Then, the plates were washed with PBS-T (0.05% Tween 20) and PBS three times for 2 min each time and incubated with 100 lL of Ó 2013 John Wiley & Sons Ltd
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anti-rabbit IgG alkaline phosphatase conjugate (final dilution 1:20 000) for 1 h. The plates were washed with PBS-T and PBS three times for 2 min each time and developed with the substrate p-nitro phenyl phosphate in substrate buffer. The optical density was measured at 405 nm using an ELISA reader (Multiskan EX Microplate Photometer; Thermo Electron Corporation). Results
The gene encoding capsid protein of MrNV was cloned in prokaryotic expression vector, pRSET B, and the clone with MCP43 gene was designated as pRMCP43 (pRSET B-M. rosenbergii nodavirus capsid protein 43 kDa). The expression of r-MCP43 was confirmed by Western blot using monoclonal antibodies raised against histidine and polyclonal antibodies raised whole MrNV (Sahul Hameed et al. 2011). The purified r-MCP43 was used to raise the antiserum in rabbit, and the efficiency of this antiserum to detect MrNV in the samples was tested by Western blot. The results revealed that the anti-rMCP43 detected MrNV in experimentally and naturally WTD-infected postlarval samples by the appearance of a distinct band of 43 kDa in Western blot (Fig. 1). The antiserum raised against r-MCP43 successfully detected MrNV in different organs such as gill tissue, pleopods, muscle and heart tissue of experimentally WTD-infected prawn by Western blot (Fig. 2). The dot blot analysis carried out using the antiserum raised against r-MCP43 was found to be capable of detecting the MrNV in WTD-infected post-larvae at 24 h post-infection (Fig. 3). The intensity of band was found to be increased during the course of infection in the post-larvae. The intensity of band was high in the samples of 5 and 7 d p.i. and moribund (Fig. 3). The dot blot analysis using the anti-rMCP43 detected MrNV in WTD-infected samples at the level of 100 pg of total protein (Fig. 4). Beyond this level, the antiserum failed to detect the MrNV in the infected samples (Fig. 4). Indirect ELISA technique was applied to quantify the 43 kDa capsid protein of MrNV in WTD-infected post-larval samples collected at different time intervals of post-infection and also in different organs of MrNV-injected adult prawn using antiserum raised against r-MCP43 which was used as a standard protein. The 43 kDa capsid protein was estimated to be about 0.57 lg per
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Figure 3 Dot blot analysis for the detection of MrNV in WTD-infected post-larvae at different time intervals (day postinfection [d p.i.]) in time course infectivity experiment. P – Positive control; N – Negative control; 1 – 1st d.p.i.; 2 – 2nd d.p.i.; 3 – 3rd d.p.i.; 4 – 5th d.p.i.; 5 – 7th d.p.i.; and 6 – Moribund. Figure 1 Western blot analysis of pRMCP43 protein of MrNV. Lane M – 100 kDa protein marker; lane 1 – Uninfected PL of Macrobrachium rosenbergii (negative control); lane 2 – Experimentally WTD-infected PL of prawn (positive control); lane 3 – Purified r-MCP43 protein; lane 4 – Induced pRMCP43-GJ1158; lane 5 – Non-induced pRMCP43GJ1158; and lane 6 – WTD-infected PL sample of M. rosenbergii (wild sample).
Figure 2 Detection of WTD-MCP43 protein by Western blot using antiserum of r-MCP43 in different organs of WTD-infected adult prawn on 3 d.p.i. Lane M – 100 kDa protein marker; lane 1 – Negative control; lane 2 – Gill tissue; lane 3 – Muscle; lane 4 – Pleopods; and lane 5 – Heart.
0.1 mg of total protein in post-larvae exposed to MrNV after 24 h p.i., and this value increased gradually during the course of infection and reached the maximum level of 1.4 lg per 0.1 mg of total protein at moribund stage (Fig. 5). The 43 kDa capsid protein of MrNV was quantified about 0.9, 1.0, 0.8 and 0.85 lg per 0.1 mg of total protein, respectively, in gill, heart, muscle and pleopods of experimentally MrNV-injected adult giant freshwater prawn. The known MrNV-positive and negative samples were coded, and the coded samples were screened for MrNV using anti-rMCP43 by Western blot, dot blot and ELISA techniques. These results were compared with that of RT-PCR to test the efficiency of antiserum raised against r-MCP43 for the detection of MrNV. The Western blot, dot blot Ó 2013 John Wiley & Sons Ltd
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Figure 4 Dot blot analysis for end-point titration to detect MrNV in WTD-infected sample. P – Positive control; N – Negative control; 1 – 10 lg of total protein; 2 – 1 lg; 3 – 100 ng; 4 – 10 ng; 5 – 1 ng; 6 – 100 pg; 7 – 10 pg.
Figure 5 Quantification of MrNV protein of WTD in PL of Macrobrachium rosenbergii at different day intervals using purified r-MCP of WTD protein as standard. PC – Positive control, NC – Negative control, 1 – 1st d.p.i.; 2 – 2nd d.p.i.; 3 – 3rd d.p.i.; 4 – 4th d.p.i., 5 – 5th d.p.i.; 6 – 6th d.p.i.; 7 – 7th d.p.i.; 8 – 8th d.p.i.; and M – Moribund.
and ELISA detected all MrNV-positive coded samples as detected by RT-PCR (Table 1). Discussion
White tail disease caused by MrNV is responsible for huge economic loss in all prawn growing countries including India. The prawn production in India has been reduced to less than 2000 tonnes from 40 000 tonnes in 2003 due to various factors including WTD. The MrNV affects early life stages such as larvae, post-larvae and juveniles, and previous reports also revealed the possibility of vertical transmission of this virus from brooder to eggs (Sahul Hameed et al. 2004b; Sudhakaran et al. 2007). Screening of brooders for MrNV and
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Table 1 Detection of MrNV in different known positive and negative coded samples using RT-PCR, Western blot, immunodot blot and ELISA assays Samples Negative control Positive control S. No.: 1 S. No.: 2 S. No.: 3 S. No.: 4 S. No.: 5 S. No.: 6 S. No.: 7 S. No.: 8 S. No.: 9 S. No.: 10
RT -PCR
ELISA
Immunodot blot
Western blot
Remarks
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XSV is essential to get disease free larvae and postlarvae. Commercial screening of the brooders by RT-PCR is costly and laborious. A simple, cost effective and moderate by sensitive diagnostic method for preliminary screening of prawn brooders would be very useful for prawn culture industry. Immunological-based diagnostic methods using the antiserum raised against whole MrNV was found to be very useful for preliminary screening. It was also found to be as sensitive as RT-PCR. Antiserum raised against purified MrNV has several limitations as observed by Romestand & Bonami (2003), Qian et al. (2006) & Sahul Hameed et al. (2011). Among these are the high cost and effort required for virus propagation which requires live animals due to lack of susceptible cell lines for this virus. White tail disease is caused by a combination of two viruses, MrNV and XSV, which have to be separated and purified for raising antiserum. Separation and purification process does not completely remove trace quantities of prawn tissue proteins that may be immunogenic. Use of recombinant DNA technology for the production of recombinant viral proteins overcomes these problems, and its purification is very simple using the histidine-tag purification system. In the present study, a bacterial host namely E. coli GJ1158 developed by Bhandari & Gowrishankar (1997) was used to produce recombinant capsid protein of MrNV (r-MCP43) with NaCl induction. This host system is very useful for the expression of cloned gene products, and the expression is under the control of osmoresponsive proU promoters. It is suitable for a variety of Ó 2013 John Wiley & Sons Ltd
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pET vectors that are used in BL21 (DE3) E. coli strains. The antiserum raised against recombinant MCP43 of MrNV was evaluated for its efficiency to detect MrNV in naturally and experimentally WTD-infected samples by different immunological methods such as Western blot, dot blot and ELISA. The anti-rMCP43 detected MrNV in different samples by Western blot as detected by the antiserum raised against MrNV (Romestand & Bonami 2003; Sahul Hameed et al. 2011). A similar study has been carried out by Yoganandhan et al. (2004) who studied the efficiency of antiserum raised against recombinant VP28 protein of white spot syndrome virus (WSSV) of shrimp to detect the WSSV in different infected samples. The anti-rMCP43 detected MrNV in gill, muscle, heart and pleopods of adult prawn, as well as post-larvae. This result revealed that all these organs including post-larvae are good sources for detection of MrNV using immunological diagnostics as observed by Sahul Hameed et al. (1998) & Yoganandhan et al. (2004) for WSSV. The dot blot assay using anti-rMCP43 was found to be capable of detecting MrNV in WTD-infected post-larvae as early as at 24 h post-infection (p.i.) as observed by Sahul Hameed et al. (2011) by Western blot and ELISA using the antiserum raised against MrNV. Similarly, Yoganandhan et al. (2004) used the antiserum raised against recombinant VP28 protein to detect WSSV in different organs of WSSV-infected shrimp by Western blot, and their results revealed the capability of this antiserum to detect WSSV within 24 h p.i. Nadala & Loh (1998) reported the detection of WSSV in experimentally infected white leg shrimp, Penaeus vannamei (Boone) at 41 h p.i. using the antiserum raised against WSSV. The antiserum raised against r-MCP43 detected MrNV in 100 pg of total protein prepared from WTD post-larvae by dot blot assay, whereas the anti-rVP28 detected WSSV in 5 ng of total haemolymph protein (Yoganandhan et al. 2004). The antiserum raised against whole WSSV detected WSSV in 800 ng of total haemolymph protein of WSSV-infected shrimp (Sahul Hameed et al. 1998). These studies clearly indicate the high efficiency of antiserum raised against recombinant viral protein for the detection of viral pathogens in comparison with antiserum raised against whole virus. In addition, production of antiserum against whole virus was proved to be difficult because of damaging of virus during
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purification, cross-reaction with host proteins and laborious for virus propagation (Nadala & Loh 2000; Chaivisuthangkura et al. 2004; Yoganandhan et al. 2004). The ELISA technique using the anti-rMCP43 was employed to quantify the capsid protein of MrNV in WTD-infected post-larvae at different day intervals after post-infection. The pure r-MCP43 was used as a standard to quantify the capsid protein of MrNV. The capsid protein was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. This indicates that the severity of WTD infection in post-larvae could be easily determined by ELISA using anti-rMCP43 and pure r-MCP43 as standard. The ELISA technique with pure r-MCP43 helped to determine the concentration of capsid protein of MrNV in different organs of MrNVinjected prawn, and the results revealed the high concentration of capsid protein in gill tissue, heart, pleopods and muscle. In the present study, we used known MrNVpositive and negative coded samples to compare our immunological diagnostic methods with RT-PCR detection system (Table 1). Results obtained were identical among Western blot, dot blot, ELISA and RT-PCR assays. These results indicate the possibility of using immunodot assay for field detection of MrNV in brooders, post-larvae and grow-out prawn which would be useful in the prawn culture industry. This kit would be economical, sensitive (equivalent to RT-PCR), rapid, very simple to perform and suitable for the development of a commercial detection kit at least for preliminary screening.
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Acknowledgements
Pillai D., Bonami J.R. & Sri Widada J. (2006) Rapid detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), the pathogenic agents of white tail disease of Macrobrachium rosenbergii (De Man), by loop-mediated isothermal amplification. Journal of Fish Diseases 29, 275–283.
The authors are grateful to the Management of C. Abdul Hakeem College, Melvisharam, India, for providing the facilities to carry out this work. This work was funded by the Department of Biotechnology, Government of India, New Delhi, India.
Puthawibool T., Senapin S., Flegel T.W. & Kiatpathomchai W. (2010) Rapid and sensitive detection of Macrobrachium rosenbergii nodavirus in giant freshwater prawns by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick. Molecular and Cellular Probes 24, 244–249.
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