Aquaculture Sci. 57（1），91－97（2009）

The Tissue Distribution of (WSSV) in Experimentally Infected Kuruma ( Japonicus) as Assessed by Quantitative Real-Time PCR

1 1 1 1 Kanako ASHIKAGA , Tomoya KONO , Kohei SONODA , Yoichi KITAO , 1 1 1, Gunimala CHAKRABORTY , Toshiaki ITAMI and Masahiro SAKAI ＊

Abstract: White spot syndrome virus (WSSV) is a highly lethal, stress dependent virus belonging to the genus Whispovirus and the family Nimaviridae. Among the various crustaceans, shrimp species are the most susceptible to WSSV infection. In the current study, a quantitative real-time PCR method was established in order to quantify the levels of WSSV in various tissues of shrimp that had been experimentally infected using the immersion bioassay. Two different concentrations of viral inoculums (high and low concentration) were used in the infection procedure. Following infection, we detected WSSV in hemolymph and other tissues including the lymphoid organ, heart, stomach and gills. Although the tissue distribution between the groups exposed to either high or low concentrations of WSSV were found to be similar, the final viral load of the tissues correlated to the concentration of WSSV used in the initial exposure. An increased viral load was confirmed in the lymphoid organ, heart, stomach and gills 10 days following infection. In contrast, an increased concentration of WSSV-DNA in hemolymph was confirmed at only 4 days post-infection.

Key words: Kuruma Shrimp; WSSV; Tissue Distribution; Real-Time PCR

tentatively named as the causative virus of this Introduction outbreak, however it was later re-designated as the penaeid rod-shaped DNA virus (PRDV) routinely suffers great pro- (Inouye et al. 1994; 1996; Momoyama et al. duction losses following disease outbreaks 1994). WSSV is a highly virulent shrimp patho- caused by viral infection (Flegel 1997; Spann gen that leads to 100% mortality within 7－10 and Lester 1997). Outbreaks of white spot days of an outbreak (Lightner 1996). In cul- syndrome virus (WSSV), yellow head virus tured penaeid , WSSV targets various (YHV) and infectious hypodermal and hemato- tissues of both ectodermal and mesodermal ori- poietic necrosis virus (IHHNV) have resulted in gins (Inouye et al 1994; Chang et al. 1996; Lo et catastrophic losses in world-wide. al. 1996). The virus is transmitted either verti- WSSV was first detected in South East Asia in cally from infected bloodstocks to larvae or hor- approximately 1992 and is currently the most izontally via water or infected animals (Lo et al. serious viral pathogen effecting commercial 1967). Hence, the viral load in infected shrimp shrimp production. It has been reported that tissue is one of the most important factors an acute- outbreak resulted in more underlying the progression and transmission of than 80% mortality in farmed kuruma shrimps the disease. Although a considerable amount (Penaeus japonicus) in Japan in 1993. The rod- of research has focused on WSSV over the past shaped nuclear virus of P. japonicus (Rv-PJ) was ten years, very few disease control strategies

Received July 29, 2008: Accepted December 15, 2008. 1 Faculty of Agriculture, University of Miyazaki, Gakuen Kibanadai nishi 1-1, Miyazaki 889-2192, Japan. ＊Corresponding author: Tel: 81-985-58-7219; Fax: 81-985-58-7219; E-mail: [email protected] 92 K. Ashikaga, T. Kono, K. Sonoda, Y. Kitao, G. Chakraborty, T. Itami and M. Sakai are currently available. tribute to the knowledge of tissue selection for Sustainability of the shrimp industry depends white spot syndrome disease diagnosis. largely on the maintenance of shrimp health through efficient disease control. Thus, early Materials and Methods disease diagnosis is crucial for the future suc- cess of shrimp farming. A wide range of diag- Viral inoculation preparation and immersion nostic methods has been developed for the bioassay detection and diagnosis of WSSV, some of which The inoculum was synthesized from WSSV are commercially available. These methods infected shrimp head that had been homog- include histological examination (Lightner 1996; enized in 3 ml of PBS. DNA was extracted Inouye et al. 1994), gene probes for use in dot- using the DNeasy Tissue kit (Qiagen,USA) and blots, in situ hybridization (Durand et al. 1996; quantified by Q-PCR to determine viral copy Nunan and Lightner 1997) and PCR (Wang et number. The Inoculum used for the immersion al. 1996; Nunan and Lightner 1997), quantitative bioassay was categorized into high (2.064× real-time PCR (Q-PCR: Durand and Lightner 1012/4 l) and low (2.064×1010/4 l) concentra- 2002) and the loop-mediated isothermal amplifi- tions, and the shrimp experimentally infected cation method (LAMP: Kono et al. 2004). via the immersion method using the two sets DNA based detection methods including of viral concentration for a period of 2.5 h. A LAMP and PCR are useful given their high sensi- total of 40 shrimps were used for each immer- tivity, however they fail to detect single copies of sion. Throughout the experiments, shrimps a viral genome in the infected tissue. Such detec- were fed with commercial pellets and sampling tion is crucial for the successful development was undertaken at 1, 4, 10 and 14 days post- of specific pathogen-free shrimp breeding pro- infection. Hemolymph, gills, lymphoid organ, grams. Therefore, quantitative analysis of viral heart and stomach tissues were collected from copy numbers by Q-PCR may provide potential the treated shrimp (5 shrimp for each tissue) to for efficient and rapid diagnosis. Q-PCR has compare differences in WSSV load. been previously utilized for the quantification of numerous human including the herpes DNA extraction simplex virus (Ryncarz et al. 1999), Epstein-Barr Various organs including heart, lymphoid virus (Kimura et al. 1999), human cytomegalovi- organ, stomach and gills were removed from the rus (Nistche et al. 1999), hepatitis B virus (Cane infected shrimp and DNA extracted using the et al. 1999) and human immunodeficiency virus DNeasy kit (Qiagen, USA). For hemolymph, (Lewin et al. 1999). DNA extraction was undertaken using the blood In the current study, we describe the genomicprep mini spin kit (GE Healthcare, UK) development of a Q-PCR method for the esti- according to the manufacturer’s instructions. mation of WSSV levels in various tissues Following extraction, the concentration of total of experimentally infected kuruma shrimp DNA in the tissue samples was estimated using (Peneaus japonicus). Among cultured peneaid a spectrophotometer. shrimps, the kuruma shrimp is the most important commercial species found in Japan Q-PCR primers and probes and Southern China. Using a known WSSV The sequence of Q-PCR primers and TaqMan inoculum, shrimps were challenged through an MGB probes (PE Applied Biosystems, USA) used immersion bioassay and the WSSV content in for the detection of WSSV were selected from different tissues at various time intervals after specific regions of the WSSV genome sequence infection determined. The aim of the present (GenBank Acc. No. AF369029). The primer pair study was to analyze the viral load in various tis- (WSSV- Fw: TGGAACAAAAGATGCTGCTCAA sues of kuruma shrimp in order to understand and WSSV-Rv: TGCGGGTCGTCGTCGAATTGT) the tissue specificity of the virus and thus, con- generated a 59 bp amplicon. The TaqMan MGB Quantiative Real-time PCR Detection in the Tissues of WSSV Infected Shrimp 93 probe (AGAATGTGGATCTTGGGC) spanned a computer, resulting in the determination of from nucleotide 212－231 of the WSSV genomic the parameter CT. The log plot of the initial sequence and was synthesized and labeled with target copy number for a set of standards (dif- the fluorescent dye 5-carboxyfluoroscein. ferent dilutions of the WSSV innocula) vs CT was determined on a straight-line plot (standard PCR conditions curve). The process of calculating CT, involved TaqMan assay was carried out using TaqMan the preparation of a standard curve and deter- Universal PCR Master Mix containing AmpliTaq mining starting copy number for samples using Gold DNA polymerase, AmpErase UNG, dNTPs GeneAmp 5700 Sequence Detection software. with dUTP and optimized buffer components A t-test was used to statistically compare the (PE Applied Biosystems, USA). A 100-ng ali- means with a defined confidence percentage of quot of DNA was added to a PCR mixture con- different WSSV virus copy number values. taining 2.25μl of each primer and 2.25μl of TaqMan MGB probe in a final volume of 25μl. Results Amplification was performed using the following program: 2 min reaction for AmpErase uracil- Specificity and sensitivity of the Q-PCR method N-glycosylase (UNG) at 50℃ and activation of A standard curve (CT) was constructed (see the AmpliTaq for 10 min at 95℃, followed by 40 Fig. 1a) in order to estimate the actual number cycles of 15 s at 95℃ and 1 min at 60℃. of infectious WSSV particles in shrimp tissues. To determine the sensitivity of the test, we found Data analysis that this method was able to detect dilutions The sensitivity of the Q-PCR assay was ranging from 1.0×104 to 1.0×1010 copies/ml of determined by testing genomic DNA pre- WSSV (Fig. 1b). pared from serial dilutions (1.0×104 to 1.0× 1010 copies/ml) of WSSV. The relationship Quantification of WSSV- DNA from shrimp fol- between viral DNA concentration and the time lowing high viral exposure following virus-exposure was established using With the exception of hemolymph, a rela- analysis of variance (ANOVA). The baseline tively high copy number of approximately 1.0 6 and threshold cycle (CT) were defined using ×10 copies/μg of WSSV DNA was observed

Fig. 1. Sensitivity test for WSDV a. Standard curve made with three replicates of each four dilutions and the value of slope, intercept and correlation. b. Amplification curve of the standard samples. 94 K. Ashikaga, T. Kono, K. Sonoda, Y. Kitao, G. Chakraborty, T. Itami and M. Sakai

Fig. 2. Concentrations of WSD viral DNA in various tissues like hemolymph, heart, lymphoid organ, stomach and gill. a. The WSDV copy number from the tissues (5 shrimp for each tissue tested) exposed in high dilution (2.064× 1012 copies / 4 l) viral inocula. b. The WSDV copy number from the tissues (5 shrimp for each tissue tested) exposed in low dilution (2.064× 1010 copies / 4 l) viral inocula. Quantiative Real-time PCR Detection in the Tissues of WSSV Infected Shrimp 95 in lymphoid, heart, stomach and gill tissue fol- WSSV DNA levels in artificially infected shrimp. lowing 1 day of infection (Fig. 2a). At 4 days Our Q-PCR amplification method was under- post infection, the viral copy number in the taken in only 1 h 40 min, with the final results hemolymph increased drastically whereas available soon after this time. This method other tissues showed a decrease in viral load. proved to be more rapid than the currently avail- After 10 days infection, the viral load was able nested or even single step PCR techniques increased in all tissues with the exception of that require 6－8 h and an electrophoresis gel hemolymph that showed a decrease in concen- set-up for result readout. tration. Among the tissues tested at 14 days In this study, we also report that the hemo- post-infection, gill and stomach contained 1.0× lymph contained the highest copy number of 108 WSSV copies/μg of DNA, which was the WSSV-DNA during the early post-infection highest concentration of viral DNA, whereas stages (approximately 4 days) at both high and heart and lymphoid tissues contained a mean of low WSSV inoculum concentrations. Whereas, 1.0×107 copies/μg of WSSV DNA. Low viral other tissues including the lymphoid organ, load was observed in the hemolymph at 14 days stomach, heart and gills showed the highest post-infection. concentration of WSSV-DNA copy at later post- infection stages (approximately 14 days). This Quantification of WSSV-DNA in shrimp tissues result indicates that once the infection is estab- during low viral exposure lished, the virus mainly concentrates in the The copy number at 1 day following infec- circulating hemolymph. As time progresses tion for both hemolymph and gill tissue was 1.0 however, infection appears to spread to other ×105 copies/μg of DNA (Fig. 2 b). Stomach, internal tissues such as the lymphoid organ and heart and lymphoid tissues contained a mean heart. Previous reports have also suggested copy number of 1.0×106 copies/μg of viral that the highest WSSV DNA copy concentra- DNA. At 4, 10 and 14 days following infection, tion is observed in hemolymph when compared the genome copy numbers were found to be to gill, muscle, pleopod and hepatopancreas between 1.0×105 to 1.0×106 copies/μg viral tissues of P. vannamei, that have been experi- DNA in gill, stomach, heart and lymphoid tis- mentally infected by viral injection (Durand and sues. The hemolymph showed a high viral DNA Lightner 2002). Our data also suggests that content at 4 days post-infection and was reduced hemolymph is not the primary site of viral rep- in concentration at 10 and 14 days post-infection. lication, but rather a medium for viral spread, as was evidenced by the decreased WSSV load Discussion at 10 and 14 days post-infection. In the cur- rent study, we also identified that the lymphoid Over the past three decades, the commercial organ and heart demonstrated the highest shrimp industry has witnessed tremendous WSSV concentration at 14 days post-infection, expansion despite the recurrence of infec- regardless of the initial viral load. Similarly, tious disease outbreaks. The risk of infectious stomach and gill tissue also demonstrated high disease spread into naïve populations has sig- WSSV concentration at 14 days post-infection. nificantly increased, as a result of the movement In contrast however, viral accumulation in these of live and frozen shrimp samples between tissues was dependent on the initial viral load countries (Lightner et al. 1996). Therefore, administered via the immersion bioassay. improvement of the currently available methods It has been reported that following oral infec- for viral detection is essential for the prevention tion in P. vannamei, the foregut and gills were of economic losses, in terms of revenue and identified as primary replication sites of WSSV resources, in the aquaculture industry. In the (Escobedo-Bonilla et al. 2007). According to current study, we applied a novel Q-PCR method Rahman et al. (2008), the number of WSSV- using Taqman probes to accurately quantify positive cells in various tissues of P. vannamei 96 K. Ashikaga, T. Kono, K. Sonoda, Y. Kitao, G. Chakraborty, T. Itami and M. Sakai was found to be significantly higher when of penaeid shrimp. Dis. Aquat. Org., 29, 205-211. injected with a more virulent strain of WSSV Durand, S., D. V. Lightner, L. M. Nunan, R. M. Redman, J. Mari and J. R. Bonami (1996) Application of gene than those injected with less virulent strains, probes as diagnostic tools for white spot baculovirus and that this increase in WSSV-positive (WSBV) of penaeid shrimp. Dis. Aquat. Org., 27, 59-66. number was more pronounced in the gill tissue. Durand, S. V. and D. V. Lightner (2002) Quantitative real Taken together, these results indicate that the time PCR for the measurement of white spot syn- drome virus in shrimp. J. Fish Dis., 25, 381-389 stomach and gills rather than other internal Escobedo-Bonilla, C. M., M. Wille, V. Alday Sanz, P. organs including the lymphoid organ and heart, Sorgeloos, M. B. Pensaert and H. J. Nauwynck (2007) are important areas of viral replication. Pathogenesis of a Thai strain of white spot syndrome In conclusion, we report the establishment virus (WSSV) in juvenile, specific pathogen-free Litopenaeus vannamei. Dis. Aquat. Organ., 74, 85-94. of a Q-PCR assay that successfully detected and Flegel, T. W. (1997) Special topic review: major viral dis- quantified WSSV in experimentally infected eases of balck tiger (Peneaus monodon) in shrimp. To the best of our knowledge, adequate Thailand. World. J. Micorbiol. Biotechnol., 13, 433-442. treatment against WSSV remains unavailable Inouye, K., S. Miwas, N. Oseko, H. Nakano, T. Kimura, K. Momoyama and M. Hiraoka (1994) Mass mortalities (Witteveldt et al. 2004) and that once introduced of cultured kuruma shrimp Peneaus japonicus in to shrimp species, spreads rapidly and uncon- Japan, 1993: Electron microscopic evidence of the trollably (Yi et al. 2003). Therefore, our Q-PCR causative virus. Fish Pathol., 29, 149-158. method may be suitable for the rapid assess- Inouye, K., K. Yamano, N. Ikeda, T. Kimura, K. Momoyama, J. Kobayashi and S. Miyajima (1996) The penaeid rod ment of disease progression in infected shrimp shaped DNA virus(PRDV) which causes penaeid acute and for the determination of WSSV content in viremia (PAV). Fish Pathol., 31, 39-45. shrimp that are resistant to WSSV infection. Tang, K. F. J. and D. V. Lightner (2000) Quantification of The present study may also provide significant white spot syndrome virus DNA through a competi- tive polymerase chain reaction. Aquaculture, 189, insight into tissue selection for WSSV diagnosis. 11-21 Finally, we hypothesize that the hemolymph Kimura, H., M. Morita, Y. Yabuta, K. Kuzushima, K. Kato, may be a novel, important marker for detection S. Kojima, T. Matsuyama and T. Morishima (1999) of WSSV during the early stages of infection, Quantitative analysis of Epstein-Barr virus load by using a real-time PCR assay. J. Clin. Microbiol., 37, while the lymphoid organ, heart, stomach and 132-136. gill tissues may be important for the detection of Lewin, S. R., M. Vesanen, L. Kostriskis, A. Hurley, M. the later stages of infection. Duran, L. Zhang, D. D. Ho and M. Markowitz (1999) Use of real time PCR and molecular beacons to detect virus replication in human immunodeficiency virus Acknowledgements type 1-infected individuals on prolonged effective anti- retroviral therapy. J. Virol., 73, 6099-6103. Funding for this research was provided by Lightner, D. V. (1996) A Handbook of Shrimp Pathology and the Research and Development Program for Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge. New Bio-industry Initiatives. Lo, C. F., J. H. Leu, C. H. Ho, C. H. Chen, S. E. Peng, Y. T. Chen, C. M. Chou, P. Y. Yen, C. J. Huang, H. Y. Chou, References C. H. Wang and G. H. Kou (1996) Detection of baculo- virus associated with white spot syndrome (WSBV) in penaeid shrimps using polymerase chain reaction. Cane, P. A., P. Cook, D. Ratcliffe, D. Mutimer and D. Pillay Dis. Aquat. Org., 25, 133-141. (1999) Use of real time PCR and fluorimetry to detect Momoyama, K., M. Hiraoka, H. Nakano, H. Kobe, K. lamivudine resistance-associated mutations in hepati- Inouye and N. G. Oseka (1994) Mass mortality of cul- tis B virus. Antimicrob. Agents Chemother., 43, tured penaeid shrimps P. japonicus in 1993: histopath- 1600-1608. ological study. Fish Pathol., 29, 141-148. Chang, P. S., C. F. Lo, Y. C. Wang and G. H. Kou (1996) Nistche, A., N. Steuer, C. A. Schmidt, O. Landt and W. Identification of white spot syndrome associated bacu- Siegert (1999) Different real time PCR formats com- lovirus WSBV target organs in the shrimp Penaeus pared for the quantitative detection of human cyto- monodon by in situ hybridization. Dis. Aquat. Org., 7, megalovirus DNA. Clin. Chem., 45, 1932-1937. 131-139. Nunan, L. M. and D. V. Lightner (1997) Development of a Durand, S., D. V. Lightner and J. R. Bonami (1997) Ultrastructural non-radioactive gene probe by PCR for detection of investigations on the white spot baculovirus (WSBV) white spot syndrome virus (WSSV). J. Virol. Methods, Quantiative Real-time PCR Detection in the Tissues of WSSV Infected Shrimp 97

63, 193-201. a PCR procedure for the detection of penaeid baculo- Rahman, M. M., M. Corteel, C. M. Escobedo-Bonilla, M. virus in shrimp. Dis. Aquat. Org., 25, 123-131. Wille, V. Alday-Sanz, M. B. Pensaert, P. Sorgeloos and Wang, C. H., C. F. Lo, J. H. Leu, C. M. Chou, P. Y. Yeh, H. H. J. Nauwynck (2008) Virulence of white spot syn- Y. Chou, M. C. Tung, C. F. Chang, M. S. Su and G. H. drome virus (WSSV) isolates may be correlated with Kou (1995) Purification and genomic analysis of bacu- the degree of replication in gills of Penaeus vannamei lovirus associated withwhite spot syndrome (WSBV) juveniles. Dis Aquat Organ., 79, 191-8. of . Dis. Aquat. Org., 23, 239-242. Ryncarz, A. J., J. Goddardet, A. Wald, M. L. Huang, B. Witteveldt, J., C. C. Cifuentes, J. M. Vlak and M. C. van Roizman and L. Corey (1999) Development of a high- Hulten (2004) Protection of Penaeus monodon against throughput quantitative assay for detecting herpes white spot syndrome virus by oral vaccination. J. simplex virus DNA in clinical samples. J. Clin. Microbiol, Virol., 78, 2057-2061. 37, 1941-1947. Wongteerasupaya, C., J. E. Vickers, S. Sriurairatana, G. L. Spann, K. M. and R. J. G. Lester (1997) Special topic review: Nash, A. Akarajamorn, V. Boonsaeng, S. Panyim, A. viral diseases of penaeid shrimp with particular refer- Tassanakajon, B. Withyachumnarnkul and T. W. Flegel ence to four viruses recently found in shrimp from (1995) A non-occluded systemic baculovirus that Queensland. World J. Microbiol. Biotech., 13, 419-426. occurs in cells of ectodermal and mesodermal origin Kono, T., R. Savan, M. Sakai and T. Itami (2004) Detection and causes high mortality in the black tiger prawn of white spot syndrome virus in shrimp by loop- Penaeus monodon. Dis. Aquat. Org., 21, 69-77. mediated isothermal amplification. J. Virol. Methods, Yi, G., J. Qian, Z. Wang and Y. Qi (2003) A phage-displayed 115, 59-65 peptide can inhibit infection by white spot syndrome Wang, S. Y., C. Hong and J. M. Lotz (1995) Development of virus of shrimp. J. Gen. Virol., 84, 2545-2553.

WSDV人工感染クルマエビの組織におけるウイルス量の解析

足利佳奈子・河野智哉・園田航平・北尾陽一 チャクラボルティー グニマラ・伊丹利明・酒井正博

WSDV は，ニマウイルス科ウィスポウイルス属に属し，海洋甲殻動物に対し非常に強い致死率を 示すウイルスである。海洋甲殻動物の中でも，エビ類は特に WSDV に対する感受性が高い。本研究 では，定量 PCR 法を用い，浸漬感染後のエビ組織における WSDV を定量した。感染後，WSDV は血 リンパ，リンパ様器官，心臓，胃および鰓において検出された。ウイルス濃度を 2 系列設け感染試験 を行った結果，濃度による組織分布に違いは見られなかったが，感染後14日目において高濃度感染区 のウイルス量は低濃度感染区と比べ顕著に増加することが確認された。また，その増加はリンパ様器 官，心臓，胃，鰓において10日目以降で確認された。一方，血リンパにおけるウイルス量の増加は感 染後 4 日目の早い段階で確認された。