Proteomic Analyses of the Shrimp White Spot Syndrome Virus

Proteomic Analyses of the Shrimp White Spot Syndrome Virus

  • Yan-wei TAN,

    Affiliation State key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences CAS, Wuhan 430071, China

  • Zheng-li SHI

    zlshi@wh.iov.cn

    Affiliation State key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences CAS, Wuhan 430071, China

Proteomic Analyses of the Shrimp White Spot Syndrome Virus

  • Yan-wei TAN, 
  • Zheng-li SHI
x

Abstract

White spot syndrome virus (WSSV), a unique member within the virus family Nimaviridae, is the most notorious aquatic virus infecting shrimp and other crustaceans and has caused enormous economic losses in the shrimp farming industry worldwide. Therefore, a comprehensive understanding of WSSV morphogenesis, structural proteins, and replication is essential for developing prevention measures of this serious parasite. The viral genome is approximately 300kb and contains more than 180 open reading frames (ORF). However, most of proteins encoded by these ORF have not been characterized. Due to the importance of WSSV structural proteins in the composition of the virion structure, infection process and interaction with host cells, knowledge of structural proteins is essential to understanding WSSV entry and infection as well as for exploring effective prevention measures. This review article summarizes mainly current investigations on WSSV structural proteins including the relative quantities, localization, function and protein-protein interactions. Traditional proteomic studies of 1D or 2D gel electrophoresis separations and mass spectrometry (MS) followed by database searches have identified a total of 39 structural proteins. Shotgun proteomics and iTRAQ were initiated to identify more structural proteins. To date, it is estimated that WSSV is assembled by at least 59 structural proteins, among them 35 are defined as the envelope fraction (including tegument proteins) and 9 as nucleocapsid proteins. Furthermore, the interaction within several major structural proteins has also been investigated. This identitification and characterization of WSSV protein components should help in the understanding of the viral assembly process and elucidate the roles of several major structural proteins.

BIOLOGICAL AND MOLECULAR CHARACTERS OF WSSV

In recent year, the annual production of shrimp in the farming industry has declined due to mass mortalities in shrimp ponds predominantly caused by viruses (33). So far, more than 20 viruses have been reported, among them, the white spot syndrome virus (WSSV) is the most devastating since it can cause up to 100% cumulative mortality within 3-7 days and leads to enormous losses to the shrimp farming industry (28). WSSV infects cultured and wild penaeid shrimp as well as most species of marine and fresh water crustaceans including crayfishes, crabs and lobsters (3, 6, 10, 15, 30). Due to its broad host range, WSSV is not only a major threat to shrimp farming, but also to the worldwide marine ecology (13).

Histopathology

Histopathological studies on WSSV infected shrimp show that the prime tissue targets are mainly those of ectodermal and mesodermal origin (31, 35, 52, 57). The initial infection starts in the stomach, gills, cuticular epidermis and the connective tissue of the hepatopancreas. At later stages, the lymphoid organ, antennal gland, muscle tissue, hematopoietic tissue, heart, hindgut and parts of the midgut also become infected. The nervous system and the compound eyes are only infected at the very late stages. The stomach, gills, cuticular epidermis, lymphoid organ, hematopoietic tissue and antennal gland are all heavily infected with WSSV at late stages of infection and become necrotic (4, 31).

Morphology

The virion of WSSV is a large, ovoid particle of about 275 nm in length and 120 nm in width, with a tail-like appendage at one end (12). So far, neither the function nor the composition of this appendage is known. The virion consists of a rod-shaped nucleocapsid with a tight-fitting capsid layer, surrounded by a loose-fitting trilaminar envelope, which consists mainly of the WSSV encoded proteins VP28 and VP19 (12, 36, 45, 48). VP28 is most likely located on the surface of the virus particle and plays a key role in WSSV infection (50). The nucleocapsid is formed by stacks of rings (about 14 in total), which are in turn formed by regular spaced globular subunits of about 8nm in diameter, arranged in two parallel rows (12, 36). The nucleocapsid contains the viral genome and consists mainly of the WSSV encoded proteins VP664, VP51C, VP60B and VP15 (42, 46, 55, 66).

Genome

The virion of WSSV contain a circular, supercoiled, double-stranded (ds) DNA genome, estimated to be ~300 kilobase pairs (kb). Three full-length genomes (307 287, 305 107 and 292 967bp in size, respectively) of WSSV isolates originating from Taiwan (WSSV-TW, GenBank accession NO. AF440570) (52), China (WSSV-CN, GenBank accession NO. AF332093) (61) and Thailand (WSSV-TH, GenBank accession NO. AF 369029) (49) have been sequenced. Between the isolates of WSSV, a few restriction fragment length polymorphisms (RFLPs) were reported, indicating the presence of some genomic variation (32, 36, 53). WSSV-TH was the first to be completely sequenced (49). Computer-assisted analysis identified 184 putative ORFs encoding proteins ranging in size from 50 to 6 077 aa in WSSV-TH genome. However, only 12 of the 184 WSSV ORFs (6%) could be assigned a putative function involved in DNA replication, nucleotide metabolism and protein modification (49).

The WSSV genome is further characterized by the presence of nine direct repeat regions with different sizes, designated homologous region (11) 1 to 9. These hrs are dispersed throughout the WSSV genome and consist of three to eight identical repeat units of 250bp or parts thereof. The hrs are largely located in intergenic regions, although several short ORFs are annotated within the WSSV hrs (49). Similar but (slightly) smaller repeat regions have been identified in ascoviruses and baculoviruses (1, 9). It was demonstrated that the hrs function as enhancers of transcription and origins of DNA replication (14, 20, 38) in Baculoviruses. However, a recent study on homologous region of Autographa californica multiple nucleopolyhedrovirus indicated that no single homologous repeat region is essential for DNA replication. Thus, the functional significance of multiple origin regions is still unclear (2).

Taxonomy

WSSV was supposed to be within the family Baculoviride. But phylogenetic analysis based on several enzyme genes including the ribonucleotide reductase large and small subunits, the protein kinases, the endonuclease, the chimeric thynmidine-hymidylate kinase and the DNA polymerase demonstrated that the WSSV was distantly related to other known viral families (8, 29, 43, 44, 47, 49, 51, 54). Based on these phylogenetic analyses and its unique morphology, WSSV had been accommodated in a new virus family, Nimaviridae, in the 8th report of International Committee on Taxonomy for Viruses (ICTV). This virus family consists of a single genus (Whispovirus) and the WSSV as its sole species so far (34, 51).

ANALYSIS OF VIRAL MAJOR STRUCTURAL PROTEINS

Identification of structural proteins by proteomic methods

With the completion of the WSSV genomic sequence, attention has been focused on the functional analysis of the encoded proteins, particularly the structural proteins. Since these proteins are the first molecules to interact with host and, therefore, play critical roles in cell targeting as well as in triggering the host defenses. Even though several major structural proteins, such as VP35, VP28, VP26, VP24, VP19 and VP15 (7, 16, 47, 48), have been successfully identified by SDS-PAGE coupled with Western blotting and/or protein N-terminal sequencing, but it is not always easy to identify every structural protein. The WSSV exhibits a large number of protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which is indicative the complexity of the virus particle.

In recent years, proteomic methods have been developed to provide powerful methods in large-scale analysis of proteins. The application of mass spectrometry followed by database searches of sequenced genomes has been proven to be a fast and sensitive for the comprehensive understanding of gene products (37). Proteins from purified virions are firstly separated by gradient SDS-PAGE and then the visible bands are excised from the gel followed by trypsin digestion and mass spectrometry to get the resulting peptide sequence data. A previous study were able to identify 18 structural proteins from WSSV by using one dimensional (1D) SDS-PAGE and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) or nano-electrospray ionization quadrupole time of flight (ESI Q-TOF) mass spectrometers (18). Later, 33 WSSV structural proteins were resolved by 2D SDS-PAGE using the online LC-ESI Q-TOF mass spectrometer and this has increased structural proteins identified to 39 by these two proteomic studies (41). Due to the low abundance of some structural proteins, the gel-based proteomic studies are not always efficient and accurate. In a recent study, in order to achieve a better understanding of the structural proteome of WSSV, shotgun proteomics using offline coupling of LC system with MALDI TOF/TOF MS/MS as a complementary and comprehensive approach to investigate the WSSV proteome. The resulting data from shotgun proteomics has identified 45 viral proteins, 13 of which are reported for the first time, the remains are identified in the previous studies (27). Therefore, the overall numbers of viral structural proteins that have been identified are 59.

Localization in virion

Up to now, the entry pathway or assembly of WSSV has not been defined due to the lack of a permissive cell culture. Therefore, a comprehensive determination of the localization of structural proteins in the virion is important to elucidate viral assembly, infection and virion morphogenesis. The conventional methods such as immunogold electron microscopy (IEM) and western blot analysis have been used to localize 14 viral proteins, including VP28, VP26, VP31, VP51C, VP36B, VP68, VP41A, VP12B, VP180, VP124, VP39, VP110 and VP24 as envelope proteins (17, 18, 23, 24, 26, 60, 63-65, 67, 68) while VP466 as nucleocapsid protein (21). Tsai et al initiated a more detailed study on WSSV structural proteins, in which they presumed there is an intermediate layer called "tegument" between envelope and nucleocapsid proteins. In their study, Triton X-100 was used in combination with various concentrations of NaCl and led to indentify 7 envelope proteins, 5 tegument proteins and 6 nucleocapsid proteins (42). Recently, a more complementary and systematic method, iTRAQ, has localized 12 novel envelope proteins and 2 novel nucleocapsid proteins (27). Further, a novel envelope protein WSV010 has been identified by shotgun proteomics approach using offline coupling LC system with MALDI-TOF/TOF MS/MS (5). In total, through different proteomic methods, 35 proteins were currently identified as envelope proteins (including tegument proteins) and 9 as nucleocapsid proteins (Table 1). The elucidation of localization of WSSV structural proteins could facilitate the investigation of the molecular mechanisms of virus assembly and virus entry.

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Table 1. The localization of structural proteins in WSSV so far characterized

Function and interaction

The nucleocapsid contains the viral genome and consists mainly of the WSSV encoded proteins VP664 and VP15 (21, 45, 46, 48). VP664, a remarkable large protein of around 664 kDa, was thought to be the major core protein responsible for the striated appearance of the nucleocapsids (21) and is quite evenly distributed at intervals on the outer surface of the nucleocapsid (42). Moreover, VP664 molecules evidently extend from the nucleocapsid to the outside surface of the tegument, which may increase the flexibility of the nucleocapsid and allows it to assume its olive-like shape in the mature virion (42). VP15, a highly basic protein with no hydrophobic regions, is a histone-like, double-stranded DNA-binding protein that tends to binds double-stranded DNA with a clear preference to supercoiled DNA, suggesting that VP15 is involved in packing the viral genome within the nucleocapsid (56).

Envelope proteins play vital roles in initiating a virus infection, including binding to receptors or penetrating into host cells by membrane fusion. VP28 is the most abundant envelope protein located on the surface of the virus particle and is supposed to play a key role in WSSV binding to shrimp cells as an attachment protein facilitating virus enter the cytoplasm (47, 62). It has been reported VP28 can bind to shrimp cells in low-pH environment and interact with host cells through PmRab7 (39). In combination with recent data, the results presented here indicated that VP28 must play a crucial role in systemic WSSV infection in shrimp. VP26 was reported to be localized in the tegument (42) and was supposed to associate loosely with both the envelope and the nucleocapsid and might function as a matrix-like linker protein between the envelope and the nucleocapsid. However, through recent immunogold labeling experiment, the "status" of VP26 was finally characterized as an envelope protein since all the gold particles localize on the outer surface of the envelope, not on the nucleocapsid or within the space between them (40). VP24 is likewise a tegument protein but we do not yet have western blotting results.

The crystalline structures of VP28 and VP26 have been determined and both of them adopt β-barrel architecture with a protruding N-terminal region. The predicted N-terminal transmembrane region of VP26 and VP28 may anchor as trimers on the viral envelope membrane, making the core β-barrel protrude outside the envelope to interact with the host receptor or to fuse with the host cell membrane for the effective transfer of the viral infection (42). Proteinprotein interactions are essential for virion morphogenesis. Using far-western and coimmunoprecipitation experiment, Xie et al. reported that VP28 interact with both VP26 and VP24 by forming a complex (59). Recently, WSV010 has been identified as a novel envelope protein and has interaction with a major viral structural protein VP24 (5). Thus, VP24 maybe also act as a linker protein for VP28, VP26 and VP24 to form a complex, which plays an important role in viral morphogenesis and infection.

To sum up, the interaction within envelope proteins of WSSV play important role in the infection process and virion morphogenesis. Tsai et al. proposed a model showing the morphogenesis of WSSV in vitro through electronic micrograph results (42). Firstly, the empty nucleocapsid forms and various envelope proteins, probably including VP28, VP26, VP24, VP31, VP36B and WSV010 assemble around it. Next, the fibrillar component (a complex of DNA and VP15 and perhaps other proteins) is folded or packed inside the nucleocapsid and the virion becomes fatter. Finally, the open end of the nucleocapsid is closed, and the virion matures. Further exploration of the biochemical interactions of WSSV structural proteins might help to elucidate the exact molecular mechanisms of virion morphogenesis.

Envelope proteins involved during infection

Sera neutralization experiment is commonly used to identify the envelope proteins involved in virus infection in vivo and in vitro. Through this method, several research groups have identified 7 envelope proteins which could delay WSSV infection significantly: VP28, VP31, VP36A, VP36B (VP281), VP466, VP68 and VP76 (19, 24, 25, 50, 58). Furthermore, their results demonstrated that the whole WSSV infection is initiated by multiple envelope proteins rather than one alone. These proteins could be the candidates for screening receptors in shrimp cell surface and are useful to discover the infection mechanism.

CONCLUDING REMARK

WSSV possess the largest genome of all animal viruses. Like other large DNA viruses such as herpesvirus, baculovirus and poxvirus, the component and structure of WSSV are much more complicated than expected. High throughput proteomics techniques proved to be very useful in studying the large DNA virus, particularly in the lack of permissive cell lines. Recently, a stable isotopic labeling, cleavable isotopecoded affinity tags (cICATs) was coupled with 2D-LC-MS/MS to quantitatively identified 33 WSSV and host proteins involved in virus infection. The advantage of this method is to identify the low quantity proteins and compare the proteins expression level during different infection stage, thus allow us to understand the whole infection process in which how the virus and host protein are involved.

However, the understanding of WSSV structure and the function of its structural proteins is still in the future. The application of classical methods such as western-blot, immunogold labeling and sera neutralization test are still needed to investigate in more detail of the function of virus genes. The other high throughput screening methods such as the two hybrid yeast systems will be used in the future to study the interaction between virus proteins, and between them and host proteins.

References

  1. 1. Bigot Y, Stasiak K, Rouleux-Bonnin F, et al. 2000. Characterization of repetitive DNA regions and methylated DNA in ascovirus genomes. J Gen Virol, 81: 3073-3082.
  2. 2. Carstens E B, Wu Y. 2007. No single homologous repeat region is essential for DNA replication of the baculovirus Autographa californica multiple nucleopolyhedrovirus. J Gen Virol, 88: 114-122.
  3. 3. Chang P S, Chen H C, Wang Y C. 1998. Detection of white spot syndrome associated baculovirus in experi-mentally infected wild shrimp, crab and lobsters by in situ hybridization. Aquaculture, 164: 233-242.
  4. 4. Chang P S, Lo C F, Wang Y C, et al. 1996. Identification of white spot syndrome associated baculovirus (WSBV) target organs in the shrimp Penaeus monodon by in situ hybridization. Dis Aquat Org, 27: 131-139.
  5. 5. Chen J, Li Z, Hew C L. 2007. Characterization of a novel envelope protein WSV010 of shrimp white spot syndrome virus and its interaction with a major viral structural protein VP24. Virology, 364: 208-213.
  6. 6. Chen L, Lo C F, Chiu Y L, et al. 2000. Experimental infection of white spot syndrome virus (WSSV) in benthic larvae of mud crab Scylla serrata. Dis Aquat Org, 40: 157-161.
  7. 7. Chen L L, Leu J H, Huang C J, et al. 2002. Identification of a nucleocapsid protein (VP35) gene of shrimp white spot syndrome virus and characterization of the motif important for targeting VP35 to the nuclei of transfected insect cells. Virology, 293: 44-53.
  8. 8. Chen L L, Wang H C, Huang C J, et al. 2002. Transcriptional analysis of the DNA polymerase gene of shrimp white spot syndrome virus. Virology, 301: 136-147.
  9. 9. Cochran M A, Faulkner P. 1983. Localization of homologous DNA sequences interspersed at five regions in the baculovirus AcMNPV genome. J Virol, 45: 961-970.
  10. 10. Corbel V, Zuprizal Z, Shi Z, et al. 2000. Experimental infection of European crustaceans with white spot syndrome virus (WSSV). J Fish Dis, 24: 377-382.
  11. 11. de Castro E, Sigrist C J A, Gattiker A, et al. 2006. ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res, 34: 362-365.
  12. 12. Durand S, Lightner D V, Redman R M, et al. 1997. Ultrastructure and morphogenesis of White spot syndrome baculovirus (WSSV). Dis Aquat Org, 29: 205-211.
  13. 13. Flegel T W. 1997. Major viral diseases of the black tiger prawn (Penaeus monodon) in Thailand. World J Microbiol Biotechnol, 13: 433-442.
  14. 14. Guarino L A, Summers M D. 1986. Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene. J Virol, 57: 563-571.
  15. 15. Hameed A S, Yoganadhan K, Sathish S, et al. 2001. White spot syndrome virus (WSSV) in two species of freshwater crabs White amanitaand P. pulvinata). Aquaculture, 201: 179-186.
  16. 16. Hameed A S, Anilkumar M, et al. 1998. Studies on the pathogenicity of systemic ectodermal and mesodermal baculovirus and its detection in shrimp by immunological methods. Aquaculture, 160: 31-45.
  17. 17. Huang C, Zhang X, Lin Q, et al. 2002. Characterization of a novel envelope protein (VP281) of shrimp white spot syndrome virus by mass spectrometry. J Gen Virol, 83: 2385-2392.
  18. 18. Huang C, Zhang X, Lin Q, et al. 2002. Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466. Mol Cell Proteomics, 1: 223-231.
  19. 19. Huang R, Xie Y, Zhang J, et al. 2005. A novel envelope protein involved in White spot syndrome virus infection. J Gen Virol, 86: 1357-1361.
  20. 20. Kool M, Van den Berg PMMM, Tramper J, et al. 1993. Location of two putative origins of DNA replication of Autographa californica nuclear polyhedrosis virus. Virology, 192: 94-101.
  21. 21. Leu J H, Tsai J M, Wang H C, et al. 2005. The unique stacked rings in the nucleocapsid of the white spot syndrome virus virion are formed by the major structural protein VP664, the largest viral structural protein ever found. J Virol, 79: 140-149.
  22. 22. Li H, Zhu Y, Xie X, et al. 2006. Identification of a novel envelope protein (VP187) gene from shrimp white spot syndrome virus. Virus Res, 115: 76-84.
  23. 23. Li L, Lin S, Yang F. 2006. Characterization of an envelope protein (VP110) of White spot syndrome virus. J Gen Virol, 87: 1909-1915.
  24. 24. Li L, Xie X, Yang F. 2005. Identification and characterization of a prawn white spot syndrome virus gene that encodes an envelope protein VP31. Virology, 340: 125-132.
  25. 25. Li L J, Yuan J F, Cai C A, et al. 2006. Multiple envelope proteins are involved in white spot syndrome virus (WSSV) infection in crayfish. Arch Virol, 151: 1309-1317.
  26. 26. Li Q, Chen Y, Yang F. 2004. Identification of a collagen-like protein gene from white spot syndrome virus. Arch Virol, 149: 215-223.
  27. 27. Li Z, Lin Q, Chen J, et al. 2007. Shotgun Identification of the Structural Proteome of Shrimp White Spot Syndrome Virus and iTRAQ Differentiation of Envelope and Nucleocapsid Subproteomes. Mol Cell Proteomics, 6: 1609-1620.
  28. 28. Lightner D V. 1996. A handbook of pathology and diagnostic procedures for diseases of penaeid shrimp. In: Special publication of the World Aquaculture Society. LA: Baton Rouge, p304.
  29. 29. Liu W J, Yu H T, Peng S E, et al. 2001. Cloning, characterization, and phylogenetic analysis of a shrimp white spot syndrome virus gene that encodes a protein kinase. Virology, 289: 362-377.
  30. 30. Lo C F, Ho C H, Peng S E, et al. 1996. White spot syndrome associated virus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Dis Aquat. Org, 27: 215-225.
  31. 31. Lo C F, Ho C H, Chen C H, et al. 1997. Detection and tissue tropism of White spot syndrome baculovirus (WSBV) in captured brooders of Penaeus monodon with a special emphasis on reproductive organs. Dis Aquat Org, 30: 53-72.
  32. 32. Lo C F, Hsu H C, Tsai M F, et al. 1999. Specific genomic DNA fragment analysis of different geographical clinical samples of shrimp white spot syndrome virus. Dis Aquat Org, 35: 175-185.
  33. 33. Lotz J M. 1997. Viruses, Biosecurity and Specific Pathogen Free Stocks In Shrimp Aquaculture. World J Microbiol Biotechnol, 13: 405-413.
  34. 34. Mayo M A. 2002. A summary of taxonomic changes recently approved by ICTV. Arch Virol, 147: 1655-1663.
  35. 35. Momoyama K, Hiraoka M, Nakano H, et al. 1994. Mass mortalities of cultured kuruma shrimp Penaeus Japonicus, in Japan in 1993: histopathological study. Fish Path, 29: 141-148.
  36. 36. Nadala E C B, Tapay L M, Loh P C. 1998. Characterization of a non-occluded baculovirus-like agent pathogenic to penaeid shrimp. Dis Aquat Org, 33: 221-229.
  37. 37. Pandey A, Mann M. 2000. Proteomics to study genes and genomes. Nature, 405: 837-846.
  38. 38. Pearson M, Bjornson R, PearsonG, et al. 1992. The Autographa californica baculovirus genome: evidence for multiple replication origins. Science, 257: 1382-1384.
  39. 39. Sritunyalucksana K, Wannapapho W, Lo C F, et al. 2006. PmRab7 is a VP28-binding protein involved in white spot syndrome virus infection in shrimp. J Virol, 80: 10734-10742.
  40. 40. Tang X, Wu J, Sivaraman J, et al. 2007. Crystal structures of major envelope proteins VP26 and VP28 from white spot syndrome virus shed light on their evolutionary relationship. J Virol, 81: 6709-6717.
  41. 41. Tsai J M, Wang H C, Leu J H, et al. 2004. Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus. J Virol, 78: 11360-11370.
  42. 42. Tsai J M, Wang H C, Leu J H, et al. 2006. Identification of the nucleocapsid, tegument, and envelope proteins of the shrimp white spot syndrome virus virion. J Virol, 80: 3021-3029.
  43. 43. Tsai J M, Wang H C, Leu J H, et al. 2000. Transcriptional analysis of the ribonucleotide reductase genes of shrimp White spot syndrome virus. Virology, 277: 92-99.
  44. 44. Tsai M F, Yu H T, Tzeng H F, et al. 2000. Identif ication and characterization of a shrimp white spot syndrome virus (WSSV) gene that encodes a novel chimeric polypeptide of cellular-type thymidine kinase and thymidylate kinase. Virology, 277: 100-110.
  45. 45. van Hulten M C, Goldbach R W, Vlak J M. 2000. Three functionally diverged major structural proteins of white spot syndrome virus evolved by gene duplication. J Gen Virol, 81: 2525-2529.
  46. 46. van Hulten M C, Reijns M, Vermeesch A M, et al. 2002. Identification of VP19 and VP15 of white spot syndrome virus (WSSV) and glycosylation status of the WSSV major structural proteins. J Gen Virol, 83: 257-265.
  47. 47. van Hulten M C, Tsai M F, Schipper C A, et al. 2000. Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions. J Gen Virol, 81: 307-316.
  48. 48. van Hulten M C, Westenberg M, Goodall S D, et al. 2000. Identification of two major virion protein genes of white spot syndrome virus of shrimp. Virology, 266: 227-236.
  49. 49. van Hulten M C, Witteveldt J, Peters S, et al. 2001. The white spot syndrome virus DNA genome sequence. Virology, 286: 7-22.
  50. 50. van Hulten M C, Witteveldt J, Snippe M, et al. 2001. White spot syndrome virus envelope protein VP28 is involved in the systemic infection of shrimp. Virology, 285: 228-233.
  51. 51. Vlak J M, Bonami J R, Flegel T W, et al. 2005. Nimaviridae. In: VIIIth Report of the International Committee on Taxonomy of Viruses (Fauquet C M, Mayo M A, Maniloff J, et al. ed), Amsterdam: Elsevier, 187-192.
  52. 52. Wang C H, Lo C F, Leu J H, et al. 1995. Purification and genomic analysis of baculovirus associated with white spot syndrome (WSBV) of Penaeus monodon. Dis Aquat Organ, 23: 239-242.
  53. 53. Wang Q, Poulos B T, Lightner D V. 2000. Protein analysis of geographic isolates of shrimp white spot syndrome virus. Arch Virol, 145: 263-274.
  54. 54. Witteveldt J, van Hulten M C, Vlak J M. 2001. Identification and phylogeny of a non-specific endonu-clease gene of white spot syndrome virus of shrimp. Virus Genes, 23: 331-237.
  55. 55. Witteveldt J, Vermeesch A M, Langenhof M, et al. 2005. Nucleocapsid protein VP15 is the basic DNA binding protein of white spot syndrome virus of shrimp. Arch Virol, 150: 1121-1333.
  56. 56. Witteveldt J, Vermeesch A M, Langenhof M, et al. 2005. Nucleocapsid protein VP15 is the basic DNA binding protein of white spot syndrome virus of shrimp. Arch Virol, 150: 1121-1133.
  57. 57. Wongteerasupaya C, Vickers J E, Sriurairatana S, et al. 1995. 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, 21: 69-77.
  58. 58. Wu W, Wang L, Zhang X. 2005. Identification of white spot syndrome virus (WSSV) envelope proteins involved in shrimp infection. Virology, 332: 578-583.
  59. 59. Xie X, Xu L, Yang F. 2006. Proteomic analysis of the major envelope and nucleocapsid proteins of white spot syndrome virus. J Virol, 80: 10615-10623.
  60. 60. Xie X, Yang F. 2006. White spot syndrome virus VP24 interacts with VP28 and is involved in virus infection. J Gen Virol, 87: 1903-1908.
  61. 61. Yang F, He J, Lin X, et al. 2001. Complete genome sequence of the shrimp white spot bacilliform virus. J Virol, 75: 11811-11820.
  62. 62. Yi G, Wang Z, Qi Y, et al. 2004. Vp28 of shrimp white spot syndrome virus is involved in the attachment and penetration into shrimp cells. J Biochem Mol Biol, 37: 726-734.
  63. 63. Zhang X, Huang C, Tang X, et al. 2004. Identification of structural proteins from shrimp white spot syndrome virus (WSSV) by 2DE-MS. Proteins, 55: 229-235.
  64. 64. Zhang X, Huang C, Xu X, et al. 2002. Identification and localization of a prawn white spot syndrome virus gene that encodes an envelope protein. J Gen Virol, 83: 1069-1674.
  65. 65. Zhang X, Huang C, Xu X, et al. 2002. Transcription and identification of an envelope protein gene (p22) from shrimp white spot syndrome virus. J Gen Virol, 83: 471-477.
  66. 66. Zhang X, Xu X, Hew C L. 2001. The structure and function of a gene encoding a basic peptide from prawn white spot syndrome virus. Virus Res, 79: 137-144.
  67. 67. Zhu Y, Xie X, Yang F. 2005. Transcription and identification of a novel envelope protein (VP124) gene of shrimp white spot syndrome virus. Virus Res, 113: 100-106.
  68. 68. Zhu Y B, Li H Y, Yang F. 2006. Identification of an envelope protein (VP39) gene from shrimp white spot syndrome virus. Arch Virol, 151: 71-82.