白斑综合症病毒（WSSV）的PCR检测条件优化及浮游动物中间宿主的调查研究
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摘要
白斑综合症病毒(white spot syndrome virus, WSSV)是迄今为止危害最为严重的一种对虾病毒。自1992年暴发以来，已在世界范围内传播开来，每年给对虾养殖业造成巨大经济损失，至今仍未得到完全控制，成为目前对虾养殖业可持续发展的主要障碍之一。十年来，人们对WSSV的传播途径进行了大量研究，确认了幼苗带病毒和水中浮游动物、池塘堤坡动物(如蟹类)和鸟粪携带病毒等的主要传播途径。近些年来，人们在生产上除采用养殖抗病力强的种类(如凡纳滨对虾)外，还采用培育健康虾苗、限制一些鲜活饵料的使用、养殖用水消毒等措施，以切断WSSV的传播途径，使得养殖生产得到了一些恢复。但是，每年全国发病的面积比例仍然很大，特别是北方地区的损失仍然相当严重。其基本原因之一就是人们对WSSV传播的某些途径仍不十分清楚，致使某个(些)传播途径仍未被有效地切断。
本研究采用灵敏、可靠的检测技术，结合具体的养殖实践，调查了WSSV在池塘养殖系统中的分布情况，并对池塘底泥中的轮虫休眠卵在传播WSSV中的作用和机制进行了初步研究，以期为今后有效杀灭WSSV提供理论依据。本文所得主要结果如下：
设计了2对引物用于检测对虾白斑综合症病毒(WSSV)，外引物用于PCR扩增，内引物用于合成探针进行斑点杂交。结果表明，外引物PCR-电泳的检出极限为1pg WSSV DNA；PCR产物经内探针斑点杂交，可检出10fg的WSSV DNA；单纯的内探针斑点杂交只能检测出1ng以上的WSSV DNA。PCR-斑点杂交的检测灵敏度比PCR-电泳高2个数量级，比单纯斑点杂交高5个数量级。Southern杂交表明，PCR-斑点杂交检测WSSV特异、可靠，该方法可用于痕量WSSV的检测。
白斑综合症病毒(WSSV)的PCR检测条件优化及浮游动物中间宿主的调查研究
PCR检测对虾白斑综合症病毒过程中用尿嗯睫一DNA糖基酶(UNG)防遗留污
染。使用UNG时，该对引物的适宜dUTP和M扩浓度分别为0.组耐和2.OmM;UNG
存在时，PCR检测WSSV DNA的最低量为1 pg，UNG的使用使PCR的检测灵敏度
降低了一个数量级;进行正常PCR扩增前，0.SU UNG可消除至少IOng含dU
的wSSV DNA的PCR扩增产物。
PCR检测攻毒鳌虾鳃样品时出现阴性，而经10一106倍稀释后的样品却呈现
阳性。结合核酸探针斑点杂交及组织切片结果，判断PCR出现了假阴性，并推
算出PCR能够成功扩增的引物与模板的比例范围为2 .43 Xl扩一2.43 X10，。。探
讨了PCR出现假阴性的原因及防范措施。
于2002年采用PCR一核酸探针斑点杂交法检测了乳山对虾养殖场1000余
份样品携带白斑综合症病毒(WSSV)的情况，结果显示:639例对虾样品阳性检
出率为26.6%;77例蟹类样品阳性检出率为18.2%;266例浮游动物样品阳性
检出率为38.3%，3月份至9月份浮游动物阳性率呈下降趋势，消毒后水体中
浮游动物的阳性率仍很高;30例螺、贝样品及22例抽滤海水样品检测均为阴
性;204例底泥样品中，阳性检出率为17.6%。
2002年在乳山养殖场采用两种围栏养殖模式养殖了中国明对虾和凡纳滨
对虾，并对其防病效果进行了比较。结果表明，中国明对虾在养殖过程中WSSv
阳性率逐渐增高，浮游动物WSSV阳性率一直很高;凡纳滨对虾在养殖过程中
wSSV阳性率有下降趋势，浮游动物wSSV阳性率较低;布围隔的防病效果好于
网围隔。
采用PCR一核酸探针斑点杂交法检测了对虾养殖池塘底泥中轮虫休眠卵及
孵出轮虫携带WSSV的情况。表面消毒后的检测结果表明，WSSV极有可能存在
于休眠卵的内部，轮虫休眠卵在对虾养殖池塘隔冬传播WSSv中可能起着重要
作用。
对采于海水养殖池塘的壶状臂尾轮虫休眠卵及孵出轮虫进行了电镜观察。
结果表明，轮虫休眠卵表面有明显的、不规则的褶皱突起，月_附着一些污染物。
轮虫细胞内线粒体、内质网密集。wSSv浸浴攻毒后轮虫出现细胞核及核仁膨大。
初步研究了池塘轮虫休眠卵及其孵出轮虫传播白斑综合症病毒(WSSV)的
白斑综合症病毒(Wssv)的PCR检测条件优化及浮游动物中间宿主的调查研究
可能性。以轮虫及其休眠卵粗提液注射淡水克氏原鳌虾，结果注射轮虫休眠卵
粗提液组未出现阳性，注射轮虫粗提液组出现1例阳性，表明轮虫有可能传播
WSSV。
White spot syndrome virus (WSSV) was the most destructive shrimp virus so far. It had spread all over the world since occurred in 1992. This virus brought great economic losses in world shrimp farming every year and has not been controlled completely till today, it was currently the main disease threat to sustainable shrimp farming. In past 10 years, much work has been done about WSSV transmission modes and several routes of infection have been found. These include not only carrier shrimp larvae, other crustaceans (e.g., crabs) and plankton in and around culture ponds, but also bird feces. Shrimp farmers have taken care to culture WSSV-resistant species (e.g., Litopenaeus vannamei), to breed WSSV-free post larvae, to restrict live food inputs, to disinfect inlet water in order to limit horizontal and vertical transmission of WSSV. Despite implementation of measures to reduce transmission, the occurrence of WSSV outbreaks was still very high in mainland China, especially in more northernly regions. This may b
e due to the fact that some major transmission routes have not yet been discovered.
WSSV distribution in shrimp pond system was studied combined with shrimp farming practice by sensitive, crediable detection techniques. Effect and mechanism of rotifer resting eggs in shrimp pond sediments in transmitting WSSV were studied primarily. These study could offer theory information to prevent WSSV transmission effectively. The primary results of the study were as follows:
Two pairs of primer were designed for white spot syndrome virus detection. The outer one was for polymerase chain reaction (PCR), the inner one was for producing probe. The results showed that the detection limit of outer primers PCR-electrophorisis was Ipg WSSV DNA, which was lOfg WSSV DNA in PCR-dot blot hybridization; Single inner primers dot blot hybridization could only detect more than Ing WSSV DNA. The sensitivity of PCR-dot blot hybridization was 102 higher than PCR-electrophorisis, 105 higher than single dot blot
hybridization. The specificity of PCR- dot blot hybridization was confirmed by Southern hybridization. The results showed that PCR-dot blot hybridization was suit for trace WSSV detection.
Uracil-DNA glycolsylase (UNO) was used to control carry-over contamination in WSSV DNA detection by PCR . Optimal dUTP and Mg2+ concentrations for this pair of primers were 0.4mM and 2.0mM respectively when UNG was present, the detection limit of WSSV DNA by PCR amplification was Ipg on this condition. UNG made this PCR sensitivity degrade one-tenth. At least lOng WSSV DNA containing dU could be abolished by 0.5U UNG prior to the normal PCR amplification .
The negative result was ocurred in WSSV injected Cambarus proclarkii detection with PCR. But, positive results were occurred when the DNA sample was diluted to 10?106 times. Together with the results of DNA probe dot blot hybridization and histopathological section, PCR false negative result could be concluded. The ratio of primer to template for sceessful amplification was 2.43 x]05 ?2.43xl010; The reason and prevent methods for PCR false negative results were
discussed.
In 2002, more than 1 000 samples were detected by PCR-DNA probe dot blot hybridization for white spot syndrome virus (WSSV) carrying status. The results showed that 26.6% was WSSV positive in 639 slirimp samples, 18.2% was positive in 77 crab samples, respectively. The WSSV positive ratio was 38.3% in 266 zooplankton samples, and the positive ratio was declined from March to September. Noticeable, the zooplankton positive ratio was still very high after water disinfection. All the 30 snail and shellfish sampbs and 22 filtered seawater samples were WSSV negative. The WSSV positive ratio was 17.6% in 204 mud samples.
In 2002, two pen-closing culturing natterns were used to culture Fenneropenaeus chinesis and Litopenaeus vannamei in Rushan shrimp farm. The effects of white spot syndrome prevention were compared. The results showed that the WSSV positive ratio was increased in Fenneropenaeus chinesis culturing process, a
本研究采用灵敏、可靠的检测技术，结合具体的养殖实践，调查了WSSV在池塘养殖系统中的分布情况，并对池塘底泥中的轮虫休眠卵在传播WSSV中的作用和机制进行了初步研究，以期为今后有效杀灭WSSV提供理论依据。本文所得主要结果如下：
设计了2对引物用于检测对虾白斑综合症病毒(WSSV)，外引物用于PCR扩增，内引物用于合成探针进行斑点杂交。结果表明，外引物PCR-电泳的检出极限为1pg WSSV DNA；PCR产物经内探针斑点杂交，可检出10fg的WSSV DNA；单纯的内探针斑点杂交只能检测出1ng以上的WSSV DNA。PCR-斑点杂交的检测灵敏度比PCR-电泳高2个数量级，比单纯斑点杂交高5个数量级。Southern杂交表明，PCR-斑点杂交检测WSSV特异、可靠，该方法可用于痕量WSSV的检测。
白斑综合症病毒(WSSV)的PCR检测条件优化及浮游动物中间宿主的调查研究
PCR检测对虾白斑综合症病毒过程中用尿嗯睫一DNA糖基酶(UNG)防遗留污
染。使用UNG时，该对引物的适宜dUTP和M扩浓度分别为0.组耐和2.OmM;UNG
存在时，PCR检测WSSV DNA的最低量为1 pg，UNG的使用使PCR的检测灵敏度
降低了一个数量级;进行正常PCR扩增前，0.SU UNG可消除至少IOng含dU
的wSSV DNA的PCR扩增产物。
PCR检测攻毒鳌虾鳃样品时出现阴性，而经10一106倍稀释后的样品却呈现
阳性。结合核酸探针斑点杂交及组织切片结果，判断PCR出现了假阴性，并推
算出PCR能够成功扩增的引物与模板的比例范围为2 .43 Xl扩一2.43 X10，。。探
讨了PCR出现假阴性的原因及防范措施。
于2002年采用PCR一核酸探针斑点杂交法检测了乳山对虾养殖场1000余
份样品携带白斑综合症病毒(WSSV)的情况，结果显示:639例对虾样品阳性检
出率为26.6%;77例蟹类样品阳性检出率为18.2%;266例浮游动物样品阳性
检出率为38.3%，3月份至9月份浮游动物阳性率呈下降趋势，消毒后水体中
浮游动物的阳性率仍很高;30例螺、贝样品及22例抽滤海水样品检测均为阴
性;204例底泥样品中，阳性检出率为17.6%。
2002年在乳山养殖场采用两种围栏养殖模式养殖了中国明对虾和凡纳滨
对虾，并对其防病效果进行了比较。结果表明，中国明对虾在养殖过程中WSSv
阳性率逐渐增高，浮游动物WSSV阳性率一直很高;凡纳滨对虾在养殖过程中
wSSV阳性率有下降趋势，浮游动物wSSV阳性率较低;布围隔的防病效果好于
网围隔。
采用PCR一核酸探针斑点杂交法检测了对虾养殖池塘底泥中轮虫休眠卵及
孵出轮虫携带WSSV的情况。表面消毒后的检测结果表明，WSSV极有可能存在
于休眠卵的内部，轮虫休眠卵在对虾养殖池塘隔冬传播WSSv中可能起着重要
作用。
对采于海水养殖池塘的壶状臂尾轮虫休眠卵及孵出轮虫进行了电镜观察。
结果表明，轮虫休眠卵表面有明显的、不规则的褶皱突起，月_附着一些污染物。
轮虫细胞内线粒体、内质网密集。wSSv浸浴攻毒后轮虫出现细胞核及核仁膨大。
初步研究了池塘轮虫休眠卵及其孵出轮虫传播白斑综合症病毒(WSSV)的
白斑综合症病毒(Wssv)的PCR检测条件优化及浮游动物中间宿主的调查研究
可能性。以轮虫及其休眠卵粗提液注射淡水克氏原鳌虾，结果注射轮虫休眠卵
粗提液组未出现阳性，注射轮虫粗提液组出现1例阳性，表明轮虫有可能传播
WSSV。
White spot syndrome virus (WSSV) was the most destructive shrimp virus so far. It had spread all over the world since occurred in 1992. This virus brought great economic losses in world shrimp farming every year and has not been controlled completely till today, it was currently the main disease threat to sustainable shrimp farming. In past 10 years, much work has been done about WSSV transmission modes and several routes of infection have been found. These include not only carrier shrimp larvae, other crustaceans (e.g., crabs) and plankton in and around culture ponds, but also bird feces. Shrimp farmers have taken care to culture WSSV-resistant species (e.g., Litopenaeus vannamei), to breed WSSV-free post larvae, to restrict live food inputs, to disinfect inlet water in order to limit horizontal and vertical transmission of WSSV. Despite implementation of measures to reduce transmission, the occurrence of WSSV outbreaks was still very high in mainland China, especially in more northernly regions. This may b
e due to the fact that some major transmission routes have not yet been discovered.
WSSV distribution in shrimp pond system was studied combined with shrimp farming practice by sensitive, crediable detection techniques. Effect and mechanism of rotifer resting eggs in shrimp pond sediments in transmitting WSSV were studied primarily. These study could offer theory information to prevent WSSV transmission effectively. The primary results of the study were as follows:
Two pairs of primer were designed for white spot syndrome virus detection. The outer one was for polymerase chain reaction (PCR), the inner one was for producing probe. The results showed that the detection limit of outer primers PCR-electrophorisis was Ipg WSSV DNA, which was lOfg WSSV DNA in PCR-dot blot hybridization; Single inner primers dot blot hybridization could only detect more than Ing WSSV DNA. The sensitivity of PCR-dot blot hybridization was 102 higher than PCR-electrophorisis, 105 higher than single dot blot
hybridization. The specificity of PCR- dot blot hybridization was confirmed by Southern hybridization. The results showed that PCR-dot blot hybridization was suit for trace WSSV detection.
Uracil-DNA glycolsylase (UNO) was used to control carry-over contamination in WSSV DNA detection by PCR . Optimal dUTP and Mg2+ concentrations for this pair of primers were 0.4mM and 2.0mM respectively when UNG was present, the detection limit of WSSV DNA by PCR amplification was Ipg on this condition. UNG made this PCR sensitivity degrade one-tenth. At least lOng WSSV DNA containing dU could be abolished by 0.5U UNG prior to the normal PCR amplification .
The negative result was ocurred in WSSV injected Cambarus proclarkii detection with PCR. But, positive results were occurred when the DNA sample was diluted to 10?106 times. Together with the results of DNA probe dot blot hybridization and histopathological section, PCR false negative result could be concluded. The ratio of primer to template for sceessful amplification was 2.43 x]05 ?2.43xl010; The reason and prevent methods for PCR false negative results were
discussed.
In 2002, more than 1 000 samples were detected by PCR-DNA probe dot blot hybridization for white spot syndrome virus (WSSV) carrying status. The results showed that 26.6% was WSSV positive in 639 slirimp samples, 18.2% was positive in 77 crab samples, respectively. The WSSV positive ratio was 38.3% in 266 zooplankton samples, and the positive ratio was declined from March to September. Noticeable, the zooplankton positive ratio was still very high after water disinfection. All the 30 snail and shellfish sampbs and 22 filtered seawater samples were WSSV negative. The WSSV positive ratio was 17.6% in 204 mud samples.
In 2002, two pen-closing culturing natterns were used to culture Fenneropenaeus chinesis and Litopenaeus vannamei in Rushan shrimp farm. The effects of white spot syndrome prevention were compared. The results showed that the WSSV positive ratio was increased in Fenneropenaeus chinesis culturing process, a
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王雷，李光友，毛远兴。口服免疫约物后中国对虾某些血淋巴因子的测定及方法研究。海洋与湖沼，1995，26(1)：34—41
夏春，黄倢。PCR法检测对虾皮下和造血器官坏死杆伏病毒。微生物学报，1999，39(2)：171—173
箫歌。中国对虾病害防治新技术—口服多糖免疫药物研制成功。海洋与湖沼，1994，25(1)：113—114
谢数涛，何建国，杨晓明，等。套式PCR检测斑节对虾白斑症病毒(WSSV)。青岛海洋大学学报，2001，31(2)：220—224
谢数涛，邱德全，何建国，等。几种理化因子对斑节对虾白斑杆状病毒(WSBV)感染力的影响。海洋科学，2000，24(3)：52—54，30
徐耀先，解梦霞，向近敏，等。病毒命名与分类系统研究进展。中国病毒学，1999，14(3)：191—204
闫冬春，黄健，董双林，等。PCR检测对虾白斑综合症病毒(WSSV)中使用UNG防遗留污染。海洋水产研究，2003，24(3)：29—33
战文斌，王远红，钟木信一，等。白斑症病毒在日本对虾体内的感染增殖。水产学报，1999，23(3)：278—282
张奇亚。国内外对虾病毒病研究综述。现代渔业信启，1995，10(10)：1—6
Chang C F, Su M S, Chen H Y, et al. Dietary β-1,3-glucan effectively improves immunity and survival of Penaeus monodon challenged with white spot syndrome virus. Fish and Shellfish Immunology, 2003, 15:297-310
Chang P S, Chen H C, Wang Y C. Detection of white spot syndrome associated baculovirus (WSBV) target organs in the Penaeus monodon by in situ hybridization. Diseases of Aquatic Organisms, 1996, 27:131-139
Chang P S, Chen H C, Wang Y C. Detection of white spot syndrome associated baculovirus in experimentally infected wild shrimp, crab and lobsters by in situ hybridization. Aquaculture, 1998, 164:233—242
Chang Y S, Lo C F, Peng S E, et al. White spot syndrome virus (WSSV) PCR-positive Artemia cysts yield PCR-negative nauplii that fail to transmit WSSV when fed to shrimp postlarve. Diseases of Aquatic Organisms, 2002, 49: 1—10
Chen L L, Wang H C, Huang C J, et al. Transcriptional analysis of the DNA polymerase gene of shrimp white spot syndrome virus. Virology, 2002, 301: 136—147
Chou H Y, Huang C Y, Wang C H, et al. Pathogenicity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan. Dis Aquat Org, 1995, 23: 165—173
Dai H P, Gao H, Zhao X Y, et al. Construction and characterization of a novel recombinant single-chain variable fragment antibody against white spot syndrome virus from shrimp. Journal of Immunological Methods, 2003, 279:267—275
Durand S V, Redman R M, Mohney L L, et al. Qualitative and quantitative sudies on the relative virus load of tails and heads of shrimp acutely infected with WSSV. Aquaculture, 2003, 216: 9—18
Guan Y Q, Yu Z M, Li C W. The effects of temperature on white spot syndrome infections in Marsupenaeus japonicus. Journal of Invertebrate Pathology, 2003, 83: 257—260
Huang C C, Song Y L. Maternal transmission of immunity to white spot syndrome associated virus (WSSV) in shrimp (Penaeus monodon). Developmental and Comparative Immunology. 1999, 23: 545—552
Inouye K, Yamano K, Ikeda N, et al. The penaeid rod-shaped DNA virus (PRDV),which causes penaeid acute viremia(PAV). Fish Pathology, 1996, 31 (1): 39—45
Kasornchandra J, Boonyaratpalin S, Itami T. Detection of wite spot syndrome in cultured penaeid shrimp in Asia:microscopic observation and polymerase chain reaction. Aquaclture, 1998, 164:243—251
Lightner D V. A handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp, section3: Virus World Aquaculture. Louisiana: Boton Rouge, 1996
Lightner D V. The Penaeid shrimp viruses TSV, IHHNV, WSSV, and YHV: current status in the Americas,available diagnostic methods,and management strategies. Journal of Applied Aquaculture, 1999, 9(2): 27—52
Liu W J, Yu H T, Peng S E, et al. Cloing, chracterization, and phylogenetic analysis of a shrimp white spot syndrome virus gene that encodes a protein kinase. Virology, 2001, 289: 362—377
Lo C F, Ho C H, Chen C H, et al. Detection of tissue tropism of white spot syndrome baculovirus (WSSV) in peneid brooders of Penaeus monodon with a special emphasis on reproductive organs. Diseases of Aquatic Organisms, 1997, 30: 53—72
Lo C F, Hsu H C, Tsai M F, et al. Specific genomic DNA fragment analysis of different geographical clinical samples of shrimp white spot syndrome virus. Dis Aquat Org, 1999, 35:175—185
Lo C F, Leu J H, Ho C H, et al. Detection of baculovirus associated with white spot syndrome (WSBV) in penaeid shrimps using polymerase chain reaction. Dis Aquat Org, 1996a, 25: 133—141
Maeda M, Kasomchandra J, ltami T, et al. Effect of various treatments on white spot syndrome virus (WSSV) from Penaeus japonicus (Japan) and P. monodon (Thailand). Fish pathology, 1998, 33(4): 381—387
Nadala E C B, Tapay L M, Lob P C. Characterization ot a non-occluded baculovirus-like agent pathogenic to penaeid shrimp. Dis Aquat Org, 1998, 33: 221—229
Nunan L M, Lightner D V. Development of a none-radio active gene probe by PCR for detection of white spot syndrome virus (WSSV). J Virol Me hods, 1997, 63(1-2): 193—201
Nunan L M, Poulos B T, Lightner D V. The detection of white spot syndrome virus(WSSV) and yellow head virus (YHV) in imported commodity shrimp. Aquaculture, 1998, 160:19—30
Peng S E, Lo C F, Lin S C, et al. The transition from pre-patent to patent infection of WSSV in P.monodon triggered by pereiopod excision. Fish Pathology, 1998, 33(4): 395—400
Rajendran K V, Vijayan K K, Santiago T C, et al. Experi nental host range and histopathology of white spot sydrome virus(WSSV) infection in shrimp, prawns, crabs and lobsters from India. Journal of Fish Diseases, 1999, 22: 183—191
Sahul Hameed A S, Murthi B L M, Rasheed M, et al. An investigation of Artemia as a possible vector for white spot syndrome virus (WSSV) transmission to Penaeus indicus. Aquaculture, 2002, 204:1—10
Sahul Hameed A S, Xavier Charles M, Anilkumar M. Tolerance of Macrobrachium rosenbergii to white spot syndrome virus. Aquaculture, 2000,183: 207—213
Soto M A, Lotz J M. Epidemiological parameters of white spot syndrome virus infections in Litopenaeus vannamei and L. setiferus. Journal of lavertebrate Pathology, 2001, 78:9—15
Sudha P M, Mohan C V, Shankar K M, et al. Relationship between white spot syndrome virus infection and clinical manifestation in Indian cultt red penaeid shrimp. Aquaculture, 1998, 167:95—101
Tang Kathy F J, Durand Stephanie V, White Brenda L, et al. Induced resistance to white spot syndrome virus infection in Penaeus stylirostris through pre-infection with infectious hypodermal and hematopoietic necrosis virus—a preliminary study. Aquaculture, 2003, 216: 19—29
Tang Kathy F J, Lightner Donald V. Quantification of white spot syndrome virus DNA through a competitive polymerase chain reaction. Aquaculture, 2000, 189: 11—21
van Hulten C W, Witteveldt J, Snippe M, et al. White spot syndrome virus envelope protein VP28 is involved in the systemic infection of shrimp. Virology, 2001, 285:228—233
van Hulten M C W, Westenberg M, Goodall S D, et al. Identification of two major virion protein genes of white spot syndrome virus of shrimp. Virology, 2000, 266: 227—236
van Hulten M C W, Witteveldt J, Peters S, el al. The white spot syndrome virus DNA genome
sequence. Virology, 2001,286(1): 7—22
Vijayan K K, Balasubramanian C P, Jithendran K P, et al. Histopathology of Y-organ in Indian white shrimp Fenneropenaeus indicus, experimentally infected with white spot syndrome virus. Aquaculture, 2003, 221: 97—106
Wang C H, Lo C F, Leu J H, et al. Purification and genomic analysis of baculovirus associated with white spot syndrome(WSBV) of Penaeus monodon. Dis Aquat Org, 1995, 23:239 —242
Wang C S, Tang K F J, Kou G H,et al. Light and electron microscopic evidence of white spot disease in the giant tiger shrimp Penaeus monodon (Fabricicus),and the kuruma shrimp Penaeus japonicus (Bate) cultured in Taiwan. Journal of Fish Diseases, 1997, 20: 323—331
Wang Q, White Brenda L, Redman Rita M, et al. Per os challenge of Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles with six geographic isolates of white spot syndrome virus. Aquaculture, 1999, 170:179—194
Wang Y G, Hassan M D, Shariff M, et al. Histopathology and cytopathology of white spot syndrome virus (WSSV) in cultured Penaeus monodon from penisular Malaysia with emphasis on pathogenesis and the mechanism of white spot formation. Diseases of Aquatic Organisms, 1999, 39: 1—11
Wang Y G, Lee K L, Najiah M, et al. A new bacterial white spot syndrome virus (BWSS) in cultured tiger shrimp Penaeus monodon and its coparision with white spot syndrome (WSS) caused by virus. Diseases of Aquatic Organisms, 2000,41: 9—18
Wang Y G, Shariff M, Sudha P M, Srinivasa Rao PS, Hassan MD, Tan LT. Managing white spot disease in shrimp. INFOFISH International, 1998, (3): 30—36
Wang Y G, Tan O L, Lee K L, Hassan M D, Shariff M (1999) Health management of shrimp during grow-out. INFOFISH International, 1999, (4): 30—36
Wang Yu-chi, Lo Chu-fang, Chang Poh-shing,et al. Experimental infection of white spot baculovirus in some cultured and wild decapods in Taiwan. Aquaculture, 1998, 164:221—231
Wongteerasupaya C, Vickers J E, Sriurairatana S, et al. A non-occluded, systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn Penaeus monodon. Dis Aquat Org, 1995, 21 : 69—77
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Yoganandhan K, Thirupathi S, Sahul Hameed A S. Biochemical, physiological and hematological changes in white spot syndrome virus-infected shrimp, Penaeus indicus. Aquaculture, 2003, 221 : 1—11
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刘萍，孔杰，石拓，等。暴发性流行病病原对中国对虾亲虾人工感染及对于代影响的PCR检测。海洋与湖沼，1999，30(2)：139—144
史成银，宋晓玲，黄倢，等。核酸斑点杂交分析法检测对虾皮下及造血组织坏死杆状病毒(HHNBV)。海洋与湖沼，1999，30(5)：486—489
宋晓玲，黄倢，王崇明，等。皮下及造血组织坏死杆状病毒对中国对虾亲虾的人工感染。水产学报，1996，20(4)：374—378
汪岷，戴继勋，张士璀，等。用ELISA检测中国对虾的白斑综合症病毒(wSSV)。海洋学报，2000，22(2)：132—136
王克行。虾病防治原理介绍。科学养鱼，1999，(9)：28—29
王雷，李光友，毛远兴。口服免疫约物后中国对虾某些血淋巴因子的测定及方法研究。海洋与湖沼，1995，26(1)：34—41
夏春，黄倢。PCR法检测对虾皮下和造血器官坏死杆伏病毒。微生物学报，1999，39(2)：171—173
箫歌。中国对虾病害防治新技术—口服多糖免疫药物研制成功。海洋与湖沼，1994，25(1)：113—114
谢数涛，何建国，杨晓明，等。套式PCR检测斑节对虾白斑症病毒(WSSV)。青岛海洋大学学报，2001，31(2)：220—224
谢数涛，邱德全，何建国，等。几种理化因子对斑节对虾白斑杆状病毒(WSBV)感染力的影响。海洋科学，2000，24(3)：52—54，30
徐耀先，解梦霞，向近敏，等。病毒命名与分类系统研究进展。中国病毒学，1999，14(3)：191—204
闫冬春，黄健，董双林，等。PCR检测对虾白斑综合症病毒(WSSV)中使用UNG防遗留污染。海洋水产研究，2003，24(3)：29—33
战文斌，王远红，钟木信一，等。白斑症病毒在日本对虾体内的感染增殖。水产学报，1999，23(3)：278—282
张奇亚。国内外对虾病毒病研究综述。现代渔业信启，1995，10(10)：1—6
Chang C F, Su M S, Chen H Y, et al. Dietary β-1,3-glucan effectively improves immunity and survival of Penaeus monodon challenged with white spot syndrome virus. Fish and Shellfish Immunology, 2003, 15:297-310
Chang P S, Chen H C, Wang Y C. Detection of white spot syndrome associated baculovirus (WSBV) target organs in the Penaeus monodon by in situ hybridization. Diseases of Aquatic Organisms, 1996, 27:131-139
Chang P S, Chen H C, Wang Y C. Detection of white spot syndrome associated baculovirus in experimentally infected wild shrimp, crab and lobsters by in situ hybridization. Aquaculture, 1998, 164:233—242
Chang Y S, Lo C F, Peng S E, et al. White spot syndrome virus (WSSV) PCR-positive Artemia cysts yield PCR-negative nauplii that fail to transmit WSSV when fed to shrimp postlarve. Diseases of Aquatic Organisms, 2002, 49: 1—10
Chen L L, Wang H C, Huang C J, et al. Transcriptional analysis of the DNA polymerase gene of shrimp white spot syndrome virus. Virology, 2002, 301: 136—147
Chou H Y, Huang C Y, Wang C H, et al. Pathogenicity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan. Dis Aquat Org, 1995, 23: 165—173
Dai H P, Gao H, Zhao X Y, et al. Construction and characterization of a novel recombinant single-chain variable fragment antibody against white spot syndrome virus from shrimp. Journal of Immunological Methods, 2003, 279:267—275
Durand S V, Redman R M, Mohney L L, et al. Qualitative and quantitative sudies on the relative virus load of tails and heads of shrimp acutely infected with WSSV. Aquaculture, 2003, 216: 9—18
Guan Y Q, Yu Z M, Li C W. The effects of temperature on white spot syndrome infections in Marsupenaeus japonicus. Journal of Invertebrate Pathology, 2003, 83: 257—260
Huang C C, Song Y L. Maternal transmission of immunity to white spot syndrome associated virus (WSSV) in shrimp (Penaeus monodon). Developmental and Comparative Immunology. 1999, 23: 545—552
Inouye K, Yamano K, Ikeda N, et al. The penaeid rod-shaped DNA virus (PRDV),which causes penaeid acute viremia(PAV). Fish Pathology, 1996, 31 (1): 39—45
Kasornchandra J, Boonyaratpalin S, Itami T. Detection of wite spot syndrome in cultured penaeid shrimp in Asia:microscopic observation and polymerase chain reaction. Aquaclture, 1998, 164:243—251
Lightner D V. A handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp, section3: Virus World Aquaculture. Louisiana: Boton Rouge, 1996
Lightner D V. The Penaeid shrimp viruses TSV, IHHNV, WSSV, and YHV: current status in the Americas,available diagnostic methods,and management strategies. Journal of Applied Aquaculture, 1999, 9(2): 27—52
Liu W J, Yu H T, Peng S E, et al. Cloing, chracterization, and phylogenetic analysis of a shrimp white spot syndrome virus gene that encodes a protein kinase. Virology, 2001, 289: 362—377
Lo C F, Ho C H, Chen C H, et al. Detection of tissue tropism of white spot syndrome baculovirus (WSSV) in peneid brooders of Penaeus monodon with a special emphasis on reproductive organs. Diseases of Aquatic Organisms, 1997, 30: 53—72
Lo C F, Hsu H C, Tsai M F, et al. Specific genomic DNA fragment analysis of different geographical clinical samples of shrimp white spot syndrome virus. Dis Aquat Org, 1999, 35:175—185
Lo C F, Leu J H, Ho C H, et al. Detection of baculovirus associated with white spot syndrome (WSBV) in penaeid shrimps using polymerase chain reaction. Dis Aquat Org, 1996a, 25: 133—141
Maeda M, Kasomchandra J, ltami T, et al. Effect of various treatments on white spot syndrome virus (WSSV) from Penaeus japonicus (Japan) and P. monodon (Thailand). Fish pathology, 1998, 33(4): 381—387
Nadala E C B, Tapay L M, Lob P C. Characterization ot a non-occluded baculovirus-like agent pathogenic to penaeid shrimp. Dis Aquat Org, 1998, 33: 221—229
Nunan L M, Lightner D V. Development of a none-radio active gene probe by PCR for detection of white spot syndrome virus (WSSV). J Virol Me hods, 1997, 63(1-2): 193—201
Nunan L M, Poulos B T, Lightner D V. The detection of white spot syndrome virus(WSSV) and yellow head virus (YHV) in imported commodity shrimp. Aquaculture, 1998, 160:19—30
Peng S E, Lo C F, Lin S C, et al. The transition from pre-patent to patent infection of WSSV in P.monodon triggered by pereiopod excision. Fish Pathology, 1998, 33(4): 395—400
Rajendran K V, Vijayan K K, Santiago T C, et al. Experi nental host range and histopathology of white spot sydrome virus(WSSV) infection in shrimp, prawns, crabs and lobsters from India. Journal of Fish Diseases, 1999, 22: 183—191
Sahul Hameed A S, Murthi B L M, Rasheed M, et al. An investigation of Artemia as a possible vector for white spot syndrome virus (WSSV) transmission to Penaeus indicus. Aquaculture, 2002, 204:1—10
Sahul Hameed A S, Xavier Charles M, Anilkumar M. Tolerance of Macrobrachium rosenbergii to white spot syndrome virus. Aquaculture, 2000,183: 207—213
Soto M A, Lotz J M. Epidemiological parameters of white spot syndrome virus infections in Litopenaeus vannamei and L. setiferus. Journal of lavertebrate Pathology, 2001, 78:9—15
Sudha P M, Mohan C V, Shankar K M, et al. Relationship between white spot syndrome virus infection and clinical manifestation in Indian cultt red penaeid shrimp. Aquaculture, 1998, 167:95—101
Tang Kathy F J, Durand Stephanie V, White Brenda L, et al. Induced resistance to white spot syndrome virus infection in Penaeus stylirostris through pre-infection with infectious hypodermal and hematopoietic necrosis virus—a preliminary study. Aquaculture, 2003, 216: 19—29
Tang Kathy F J, Lightner Donald V. Quantification of white spot syndrome virus DNA through a competitive polymerase chain reaction. Aquaculture, 2000, 189: 11—21
van Hulten C W, Witteveldt J, Snippe M, et al. White spot syndrome virus envelope protein VP28 is involved in the systemic infection of shrimp. Virology, 2001, 285:228—233
van Hulten M C W, Westenberg M, Goodall S D, et al. Identification of two major virion protein genes of white spot syndrome virus of shrimp. Virology, 2000, 266: 227—236
van Hulten M C W, Witteveldt J, Peters S, el al. The white spot syndrome virus DNA genome
sequence. Virology, 2001,286(1): 7—22
Vijayan K K, Balasubramanian C P, Jithendran K P, et al. Histopathology of Y-organ in Indian white shrimp Fenneropenaeus indicus, experimentally infected with white spot syndrome virus. Aquaculture, 2003, 221: 97—106
Wang C H, Lo C F, Leu J H, et al. Purification and genomic analysis of baculovirus associated with white spot syndrome(WSBV) of Penaeus monodon. Dis Aquat Org, 1995, 23:239 —242
Wang C S, Tang K F J, Kou G H,et al. Light and electron microscopic evidence of white spot disease in the giant tiger shrimp Penaeus monodon (Fabricicus),and the kuruma shrimp Penaeus japonicus (Bate) cultured in Taiwan. Journal of Fish Diseases, 1997, 20: 323—331
Wang Q, White Brenda L, Redman Rita M, et al. Per os challenge of Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles with six geographic isolates of white spot syndrome virus. Aquaculture, 1999, 170:179—194
Wang Y G, Hassan M D, Shariff M, et al. Histopathology and cytopathology of white spot syndrome virus (WSSV) in cultured Penaeus monodon from penisular Malaysia with emphasis on pathogenesis and the mechanism of white spot formation. Diseases of Aquatic Organisms, 1999, 39: 1—11
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