VetoFish - Manuel des tests de diagnostic pour les animaux aquatiques

Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 4.1.4.
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CHAPTER 4.1.4.

TETRAHEDRAL BACULOVIROSIS
(Baculovirus penaei)

SUMMARY
Baculovirus penaei (BP) is an occluded baculo-like virus that contains double-stranded DNA as its nucleic acid type (2, 3, 11, 12, 16, 25, 32). Although BP was not listed with the baculoviruses in the Seventh Report of International Committee on Taxomony of Viruses (ICTV) (34), it had been cited and recognised as a possible member of the nuclear polyhedrosis genus of the Baculoviridae in the Fifth Report of the ICTV (14). Furthermore, Penaeus monodon-type baculovirus (MBV), a related nuclear polyhedrosis virus of Eastern Hemisphere penaeid shrimp, was listed as a possible member of this group in the Seventh Report of the ICTV (34). According to ICTV guidelines (14, 26, 34), BP was designated PvSNPV (for singly enveloped nuclear polyhedrosis virus from P. vannamei, the most characterised geographical strain of BP from P. vannamei) (2). Although PvSNPV may be the most correct name for BP, the term BP will be used to designate this virus (and its closely strains) in this Manual.

BP is considered to be a potentially serious pathogen in the larval, postlarval, and early juvenile stages of host shrimps. BP possesses a wide geographical distribution and diverse host range, and multiple strains of the virus have been documented (6, 10, 13, 17-19, 21, 29-31). Infections by BP are characterised by the presence of prominent, tetrahedral intranuclear occlusion bodies, which are referred to as polyhedral occlusion bodies or polyhedral inclusion bodies, in affected epithelial cells of the hepatopancreas and midgut, or free within lysed cell debris in the faeces (3-5, 21, 22). Crowding, chemical stress, or environmental stress may enhance the pathogenicity and increase the prevalence of BP in its hosts (11). Infection by BP is exclusively by the oral route in which cannibalism and faecal-oral contamination are the principal mechanisms of transmission (15, 16, 21, 22, 27, 29).

BP has a widespread distribution in cultured and wild penaeid shrimps in North and South America, but its geographical distribution is limited to the Western Hemisphere (2-5, 10, 20-24, 27, 35). However, within the Americas and Hawaii, multiple geographical strains of BP exist, and some of these may be distinguished by the size of the virion or by molecular methods (9, 13, 21, 24).

Several surveillance methods are available for use in certification of the BP infection status of shrimp stocks. The simplest method is based on the microscopic demonstration of the characteristic occlusion bodies produced by the virus. With direct microscopy, characteristic tetrahedral occlusion bodies are demonstrated in wet-mounts of whole larvae, of excised portions of the hepatopancreas from postlarvae or older shrimp, or of faeces from large juvenile to adult shrimp. Wet-mount examination of the faeces of adult broodstock for characteristic occlusion bodies may be used as a simple nonlethal method to detect carriers with moderate or heavier infections (3-5, 20, 21). However, the wet-mount method may not detect developing, low-grade, or low prevalence BP infections. Histology of fixed specimens may also be used for surveillance (1, 21). Routine histology provides a positive diagnosis of BP infection when characteristic tetrahedral occlusion bodies are demonstrated in hypertrophied nuclei of mucosal epithelial cells of the hepatopancreas or midgut (3-5, 20, 21). Molecular detection methods for BP, (gene probes applicable to in-situ hybridisation assays and polymerase chain reaction methods) are also available and provide a satisfactory method for surveillance applications (3, 7-9, 13, 21, 22, 35).

Because BP is transmitted from adults to their offspring by faecal contamination of the spawned eggs, prevention of infection in hatcheries may be achieved by taking steps to eliminate faecal contamination of spawned eggs and larvae thorough the use of disinfectants, drying, and other routine sanitation methods with tanks and equipment (18), and by direct decontamination of spawns by washing nauplii or eggs with formalin, iodophores, and clean sea water (28).

DIAGNOSTIC PROCEDURES
Infection of the hepatopancreas by the nuclear polyhedrosis virus Baculovirus penaei (BP) is among the most readily diagnosable disease of the penaeid shrimps. The occlusion bodies formed by the virus are very conspicuous and easily demonstrated by direct light microscopy with fresh specimens or by routine histological methods with fixed specimens. Direct microscopic methods are most suitable for the postlarvae stages, which are commonly moved in regional and international commerce (3-5, 20-22). Highly sensitive molecular methods for BP are also available and provide alternative methods for surveillance applications, especially for nonlethal testing of broodstock (21, 22, 35). Hence, there are several options to choose from as surveillance methods for certification of the BP-infection status of shrimp stocks.

The methods currently available for surveillance, detection, and diagnosis of BP are listed in Table 1. The designations used in the Table indicate: - = the method is presently unavailable or unsuitable; ? = the method is available but untested; + = the method has application in some situations, but cost, accuracy, or other factors severely limit its application; ++ = the method is a standard method with good diagnostic sensitivity and specificity; and +++ = the method is the recommended method for reasons of availability, utility, and diagnostic specificity and sensitivity.

Sampling procedures: see Chapter I.3.

Table 1. Surveillance, detection and diagnostic methods for the nuclear polyhedrosis virus BP.

Method

Screening

Presumptive

Confirmatory

Larvae

PLs

Juveniles

Adults

Gross signs

-

-

-

-

+

-

Direct BF/LM

+++

+++

+

++

+++

+++

Histopathology

+++

+++

++

++

+++

+++

Bioassay

-

-

-

-

+

+

Transmission EM

-

-

-

-

+

+

Antibody-based methods

-

-

-

-

-

-

DNA probes - dot-blot

-

-

-

-

?

?

DNA probes - in situ

-

-

-

-

+++

+++

PCR

+++

+++

+++

+++

+++

+++

1.	Standard Screening Methods for BP
	1.1.	Direct microscopic methods
		.	1.1.1. Wet-mount of fresh tissue
			Diagnosis of BP infections is made by the demonstration of single or multiple tetrahedral occlusion bodies in epithelial cell nuclei in squash preparations of hepatopancreas or midgut examined by phase-contrast or bright-field microscopy. Occlusion bodies are tetrahedral or pyramidal in three-dimensional form, and range in size from less than 0.1 µm to nearly 20 µm from pyramidal base to peak, with a modal, vertical length of 8 µm. In some publications, the occlusion bodies of BP are referred to as PIBs (polyhedral inclusion bodies) (3-5, 11, 12, 16, 20-22).
		.	1.1.2. Faecal examination technique
			This nonlethal method may be used to screen for carriers of BP. The method can be applied to juvenile or older shrimp, and it is perhaps most useful as a nonlethal method for screening valuable broodstock. Faecal samples from shrimp to be tested may be obtained by placing the shrimp in an aquarium, spawning tank, or other suitable tanks for a few hours until faecal strands are present on the tank bottom. The faecal strands are best collected using a clear plastic siphon hose (an air line fitted with a section of plastic pipette as a tip is ideal) and placed in a beaker, cup, or suitable container. The faecal strands may be made into wet-mounts and examined directly for occlusion bodies. BP occlusion bodies are prominent, refractive tetrahedrons that range from just resolvable to nearly 20 µm in height (3-5, 20-22).
			Collected faeces may also be used as the sample for nonlethal testing for BP by polymerase chain reaction (PCR). PCR will provide greater diagnostic sensitivity for low-grade infections than will direct microscopic examination (3, 21).
	1.2.	Histological methods
		See Section 2.2.
	1.3.	Polymerase chain reaction procedure
		PCR methods have been developed for BP. Primer sets designed from certain segments of the BP genome are unique and provide positive test results only in samples infected with particular stains of this virus (3, 35).
		Substances in the hepatopancreas and faeces of shrimp have been found to inhibit the DNA polymerase used in the PCR assay. Therefore, DNA extraction is required before PCR can be successfully used to detect BP (35).
		.	1.3.1. DNA extraction
			DNA extraction kits are convenient and commercially available. Otherwise, a suitable DNA extraction procedure is as follows:
			i)	A sample of faeces or hepatopancreas is added to digestion buffer (~1:10 ratio of sample:buffer in up to 400 µl of buffer) containing 50 mM KCl, 10 mM Tris/HCl, pH 8.3, 0.1 mg/ml gelatin, 0.45% Nonidet P-40, 0.45% Tween 20, and 80 µg/ml proteinase K, crushed, and dispersed with a wooden toothpick or a pipette tip.
			ii)	After dispersing the sample in the digestion buffer, heat to 60°C for 1 hour and then to 95°C for 10 minutes.
			iii)	Centrifuge at 12,000 g for 2 minutes, transfer the supernatant fluid to a new tube, and store on ice.
			iv)	Remove 50 µl of the digested sample and dilute with 150 µl of dilution buffer (10 mM Tris/HCl, pH 8.0, 0.1 mM ethylene diamine tetra-acetic acid [EDTA]) and extract with 200 µl of phenol/isoamyl alcohol/chloroform (PIC) (25/1/24).
			v)	After vortexing the sample for 5 seconds, leave the tube for 5 minutes and then centrifuge at 12,000 g for 2 minutes.
			vi)	Remove 160 µl of the aqueous (upper) phase and transfer to a new microcentrifuge tube.
			vii)	The extraction step may be repeated if necessary.
			viii)	Precipitate the DNA by adding 20 µg of glycogen (1 µl of a 20 mg/ml stock), 65 µl of a 7.5 M ammonium acetate and 390 µl of ethanol, store at -20°C for >1 hour, and then pellet the DNA by centrifugation at 12,000 g for 5 minutes.
			ix)	Rinse the DNA pellet with 200 µl of 70% ethanol to remove residual ammonium acetate, dry, and then dissolve the DNA pellet in 30 µl of dilution buffer or distilled water prior to adding the sample (template) to the PCR reaction mixture and beginning the PCR.
		.	1.3.2. BP PCR method
			Three forward and three reverse primers selected from an ~1430 base pair (bp) segment of the BP polyhedrin gene have been reported by Wang et al. (35).
			The sequences for these primers are:

BPA	5'-GAT-CTG-CAA-GAG-GAC-AAA-CC-3'	Temperature 61°C
BPB	5'-ATC-GCT-AAG-CTC-TGG-CAT-CC-3'	Temperature 64°C
BPD	5'-TGT-TCT-CAG-CCA-ATA-CAT-CG-3'	Temperature 62°C
BPE	5'-TAC-ATC-TTG-GAT-GCC-TCT-GC-3'	Temperature 63°C
BPF	5'-TAC-CCT-GCA-TTC-CTT-GTC-GC-3'	Temperature 68°C
BPG	5'-ATC-CTG-TTT-CCA-AGC-TCT-GC-3'	Temperature 64°C

			The combinations of these primers amplify segments from BP template DNA of: BPA/BPF - 196 bp; BPA/BPB - 560 bp; BPA/BPG - 933 bp; BPD/BPB - 207 bp; BPD/BPG - 580 bp; and BPE/BPG - 221 bp.
			The following PCR procedure was adapted from Wang et al. (35):
			i)	For BP PCR, the DNA in each extracted sample (Section 1.3.1.) is denatured by heating in a boiling water bath for 3 minutes followed by quick chilling in ice-water.
			ii)	25 µl reaction mixture containing 5 mM of each primer, 1.5 mM MgCl₂, and 0.5-1 unit of DNA polymerase is added.
			iii)	After heating the reaction mixture for 3 minutes at 95°C, 30 PCR cycles (a DNA melting step at 94°C, a primer annealing step at 60°C, and an elongation step at 72°C) are performed followed by an elongation step of 5 minutes at 72°C.
			iv)	The resultant PCR products may be compared with molecular standards by 2% agarose gel electrophoresis or assayed for with a specific DNA probe for the fragment following a Southern transfer.
			v)	The following controls should be included in every PCR assay for BP: a known negative tissue or faecal sample; a known positive tissue or faecal sample (this can be the DNA clone from which a specific set of primers was designed); and a 'no- template' control.
2.	Diagnostic Methods for Confirmatory Tests
	Confirmation of infection by BP may be accomplished with any of the methods listed in Section 1 (i.e. wet-mounts of hepatopancreas tissue squashes or of faecal strands, or by PCR). The other methods available for confirmatory diagnosis of BP include: autofluorescence with phloxine, and routine histological methods (1, 3-5, 20-22, 33).
	2.1.	Autofluorescence method with phloxine stain
		Another method for detecting BP occlusion bodies is based on the fluorescence of phloxine-stained occlusion bodies (3, 21, 33). Aqueous 0.001% phloxine may be added to tissue squash preparations to make wet-mounts of hepatopancreas or faeces for direct examination. Histological sections stained with routine haematoxylin and eosin (H& E) containing 0.005% phloxine, are also suitable for this procedure. BP occlusions in wet-mounts of tissue squashes, in faeces, or in histological sections fluoresce bright yellow-green against a pale green background under epi-fluorescence (barrier filter of 0-515 nm and a 490 nm exciter filter). Other objects in the tissues and insect baculovirus occlusion bodies do not fluoresce with this method. Hence, the method can provide a rapid and specific diagnosis.
	2.2.	Histological methods
		Histology may be used to provide a definitive diagnosis of BP infection. Because 10% buffered formalin and other fixatives provide, at best, only fair fixation of the shrimp hepatopancreas (the principal target organ for BP and other baculo-like virus infections of penaeid shrimp), the use of Davidson's fixative (containing 33% ethyl alcohol [95%], 20% formalin [approximately 37% formaldehyde], 11.5% glacial acetic acid, and 33.5% distilled or tap water) is highly recommended for all routine histological studies of shrimp (1, 3, 21). To obtain the best results, dead shrimp should not be used. Only live, moribund, or compromised shrimp should be selected for fixation and histological examination. Selected shrimp are killed by injection of fixative directly into the hepatopancreas; the cuticle over the cephalothorax and abdomen just lateral to the dorsal midline is opened with fine-pointed surgical scissors to enhance fixative penetration (the abdomen may be removed and discarded), the whole shrimp (or cephalothorax less the abdomen) is immersed in fixative for 24-48 hours, and then transferred to 70% ethyl alcohol for storage. After transfer to 70% ethyl alcohol, fixed specimens may be transported by wrapping in cloth or a paper towel saturated with 70% ethyl alcohol and packed in leak-proof plastic bags. To begin histological processing, fixed shrimp are 'cut-in' (see ref. 1 for a photographic guide to this procedure) to facilitate eventual sectioning of the hepatopancreas and midgut. After dehydration, the specimens are embedded in paraffin and sections of 5-7 µm thickness are cut. Routine histological stains such as Mayer-Bennett's or Harris H& E, Giemsa stains, and Gram tissue-staining methods may be used for the demonstration of pathognomonic (for BP) tetrahedral occlusion bodies in hepatopancreatocytes, gut epithelial cells, or gut lumen (3, 4, 6, 21). Typically, BP-infected hepatopancreatic (or occasionally midgut) cells will present markedly hypertrophied nuclei with single or, more often, multiple occlusion bodies, chromatin diminution and margination. Occlusion bodies may be stained bright red with H& E stains, and intensely, but variably, with Gram's tissue stains. Brown and Brenn's histological Gram stain, although not specific for baculovirus occlusion bodies, tends to stain occlusions more intensely (either red or purple, depending on section thickness, time of decolouration, etc.) than the surrounding tissue, aiding in demonstrating their presence in low-grade infections (3-5, 20-22).
	2.3.	Molecular methods
		Nonradioactive DIG-labelled gene probes to BP have been developed and some are commercially available (3, 7-9, 21, 22, 25). Gene probe and PCR methods may provide greater diagnostic sensitivity in detecting low-grade infections than do more traditional wet-mount or histological techniques. Furthermore, the PCR method (see Section 1.3.) has the added advantage of being applicable to nonlethal testing of faecal samples collected from valuable broodstock shrimp.
		DIG-labelled DNA probes for representative strains of BP are commercially available as ShrimProbeTM kits from DiagXotics (Wilton, Connecticut, USA). The probes are labelled with a nonradioactive label, digoxigenin-11-dUTP (DIG). These probes only work well with the in-situ hybridisation method with histological sections because there are substances present in the hepatopancreas and faeces of shrimp that provide both false-positive and false-negative results with samples that are blotted directly and not extracted prior to probing.
		.	2.3.1. Dot-blot hybridisation procedure for BP
			While specific DNA probes for BP are available, their use in dot-blot hybridisation procedures is not recommended for most routine diagnostic applications. Pigments present in the hepatopancreas leave a coloured spot on the hybridisation membrane that can result in the masking of a positive test or in the false interpretation of a negative test. Likewise, bits of chitin (which nonspecifically bind DNA probes), pigments, and other materials present in the faecal sample may also result in false-positive or false-negative dot-blot hybridisation tests. Extraction of DNA from the hepatopancreas or faeces prior to blotting or the use of chemiluminescent or radioactively labelled probes may circumvent these problems, but the adequacy of other test methods (i.e. direct wet-mounts, histology, or PCR) has not indicated a need for the further refinement and application of the dot-blot method (21).
		.	2.3.2. *In-situ* hybridisation procedure
			The in-situ hybridisation protocol given in detail in Section 1.2. of Chapter 4.1.6. Infectious hypodermal and haematopoietic necrosis virus (IHHNV), uses the GeniusTM System developed by Boehringer Mannheim Biochemicals (now Roche Diagnostics) and was adapted from the Boehringer Mannheim's Nonradioactive In Situ Hybridization Application Manual. An additional step is required for the in-situ hybridisation test for BP. Follow steps i-vi of Section 1.2., in-situ hybridisation procedure for IHHNV, in Chapter 4.1.6. For BP substitute the following modifications to step vii before proceeding with steps viii-xvii as given for IHHNV:
			i)	(Modified step vii): Boil the DIG-labelled probe for 10 minutes and quench on ice; spin briefly in the cold (~4-10°C) using a refrigerated centrifuge or a chilled microcentrifuge to bring all the liquid down to the base of the microcentrifuge tube; keep on ice. Dilute the probe to 50 ng/ml in prehybridisation solution and cover the tissue with 500 µl of the solution. Denature the double-stranded viral DNA in the tissue of the histological section by placing the slides on a 85°C heat block (or on aluminium foil that is placed over a boiling water bath) for 6-10 minutes. Quench the slides on ice for 5 minutes. Incubate the slides overnight at 42°C in a humid chamber. Drain the fluid on to blotting paper. During this incubation step, keep the wash buffers at 37°C to prewarm them.
			ii)	Proceed with steps viii-xvii as given for IHHNV.
				With DIG-labelled probes, accumulations of BP viral DNA within infected cell nuclei, in cytoplasmic phagosomes, or in necrotic tissue debris are stained blue to a dark blue-black. Although they contain virus, occlusion bodies do not normally react with DIG-labelled DNA probes because the occlusion body protein crystalline matrix does not permit penetration of the probe (21).

1.	Bell T.A. & Lightner D.V. (1988). A Handbook of Normal Shrimp Histology. Special Publication No. 1. World Aquaculture Society, Baton Rouge, Louisiana, USA, 114 pp.
2.	Bonami J.R., Bruce L.D., Poulos B.T., Mari J. & Lightner D.V. (1995). Partial characterisation and cloning of the genome of PvSNPV (= BP-type virus) pathogenic for Penaeus vannamei. Dis. Aquat. Org., 23, 59-66.
3.	Bondad-Reantaso M.G., McGladdery S.E., East I., Subasinghe R.P., eds. (2001). Asia Diagnostic Guide to Aquatic Animal Diseases. FAO Fisheries Technical Paper 402, Supplement 2. Rome, FAO, 240 pp.
4.	Brock J.A. & Lightner D.V. (1990). Diseases of crustacea. Diseases caused by microorganisms. In: Diseases of Marine Animals, Vol. III, Kinne O., ed. Biologische Anstalt Helgoland, Hamburg, Germany, 245-349.
5.	Brock J.A. & Main K. (1994). A Guide to the Common Problems and Diseases of Cultured Penaeus vannamei. Published by the Oceanic Institute, Makapuu Point, P.O. Box 25280, Honolulu, Hawaii, USA.
6.	Brock J.A., Nakagawa L.K., Van Campen H., Hayashi T. & Teruya S. (1986). A record of Baculovirus penaei from Penaeus marginatus Randall in Hawaii. J. Fish Dis., 9, 353-355.
7.	Bruce L.D., Lightner D.V., Redman R.M. & Stuck K.C. (1994). Application of traditional and molecular detection methods to experimental studies on the development of Baculovirus penaei (BP) infections in larval Penaeus vannamei. J. Aquat. Anim. Health, 6, 355-359.
8.	Bruce L.D., Redman R.M. & Lightner D.V. (1994). Application of gene probes to determine target organs of a penaeid shrimp baculovirus using in situ hybridization. Aquaculture, 120, 45-51.
9.	Bruce L.D., Redman R.M., Lightner D.V. & Bonami J.R. (1993). Application of gene probes to detect a penaeid shrimp baculovirus in fixed tissue using in situ hybridization. Dis. Aquat. Org., 17, 215-221.
10.	Bueno S.L.S., Nascimento R.M. & Nascimento I. (1990). Baculovirus penaei infection in Penaeus subtilis: A new host and a new geographic range of the disease. J. World Aquaculture Soc., 21, 235-237.
11.	Couch J.A. (1974). An enzootic nuclear polyhedrosis virus of pink shrimp: ultrastructure, prevalence, and enhancement. J. Invertebr. Pathol., 24, 311-331.
12.	Couch J.A. (1974). Free and occluded virus similar to Baculovirus in hepatopancreas of pink shrimp. Nature, 247 (5438), 229-231.
13.	Durand S., Lightner D.V. & Bonami J.R. (1998). Differentiation of BP-type baculovirus strains using in situ hybridization. Dis. Aquat. Org., 32, 237-239.
14.	Francki R.I.B., Fauquet C.M., Knudson D.L. & Brown F. (1991). Classification and Nomenclature of Viruses. Fifth Report of the International Committee on Taxonomy of Viruses. Arch. Virol. (Suppl. 2). Springer-Verlag, Vienna, Austria and New York, USA, 450 pp.
15.	Hammer H.S., Stuck K.C. & Overstreet R.M. (1998). Infectivity and pathogenicity of Baculorirus penaei (BP) in cultured larval and postlarval Pacific white shrimp, Penaeus vannamei, related to the stage of viral development. J. Invertebr. Pathol., 72, 38-43.
16.	Johnson P.T. & Lightner D.V. (1988). Rod-shaped nuclear viruses of crustaceans: gut-infecting species. Dis. Aquat. Org., 5, 123-141.
17.	Le Blanc B.D. & Overstreet R.M. (1990). Prevalence of Baculovirus penaei in experimentally infected white shrimp (Penaeus vannamei) relative to age. Aquaculture, 87, 237-242.
18.	Le Blanc B.D. & Overstreet R.M. (1991). Effect of dessication, pH, heat and ultraviolet irridation on viability of Baculovirus penaei. J. Invertebr. Pathol., 57, 277-286.
19.	Le Blanc B.D., Overstreet R.M. & Lotz J.M. (1991). Relative susceptibility of Penaeus aztecus to Baculovirus penaei. J. World Aquaculture Soc., 22, 173-177.
20.	Lightner D.V. (1988). Diseases of cultured penaeid shrimp and prawns. In: Disease Diagnosis and Control in North American Marine Aquaculture, Sindermann C.J. & Lightner D.V., eds. Elsevier, Amsterdam, The Netherlands, 8-127.
21.	Lightner D.V. (Ed.) (1996). A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge, Louisiana, USA. 304 pp.
22.	Lightner D.V & Redman R.M. (1998). Shrimp diseases and current diagnostic methods. Aquaculture, 164, 201-220.
23.	Lightner D.V., Redman R.M. & Almada Ruiz E.A. (1989). Baculovirus penaei in Penaeus stylirostris (Crustacea: Decapoda) cultured in Mexico: unique cytopathology and new geographic record. J. Invertebr. Pathol., 53, 137-139.
24.	Lightner D.V., Redman R.M., Williams R.R., Mohney L.L., Clerx J.P.M., Bell T.A. & Brock J.A. (1985). Recent advances in penaeid virus disease investigations. J. World Mariculture Soc., 16, 267-274.
25.	Machado C.R., Bueno S.L. de S. & Menck C.F.M. (1995). Cloning shrimp Baculovirus penaei DNA and hybridization comparison with Autographa californica nuclear polyhedrosis virus. Rev. Brasil. Genet. (Brazil. J. Genetics), 18, 1-6.
26.	Murphy F.A., Fauquet C.M., Mayo M.A., Jarvis A.W., Ghabrial S.A., Summers M.D., Martelli G.P. & Bishop D.H.L. (1995). Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses. Arch. Virol. (Suppl. 10). Springer-Verlag, Vienna, Austria and New York, USA. 586 pp.
27.	Overstreet R.M., Stuck K.C., Krol R.A. & Hawkins W.E. (1988). Experimental infections with Baculovirus penaei in the white shrimp Penaeus vannamei as a bioassay. J. World Aquaculture Soc., 19, 175-187.
28.	Sano T. & Momoyama K. (1992). Baculovirus infection of penaeid shrimp in Japan. In: Diseases of Cultured Penaeid Shrimp in Asia and the United States, Fulks W. & Main K., eds. The Oceanic Institute, Makapuu Point, Honolulu, Hawaii, USA, 169-174.
29.	Stuck K.C. & Overstreet R.M. (1994). Effect of Baculovirus penaei on growth and survival of experimentally infected postlarvae of the Pacific white shrimp, Penaeus vannamei. J. Invertebr. Pathol., 64, 18-25.
30.	Stuck K.C., Stuck L.M., Overstreet R.M. & Wang S.Y. (1996). Relationship between BP (Baculovirus penaei) and energy reserves in larval and postlarval Pacific white shrimp Penaeus vannamei. Dis. Aquat. Org., 24, 191-198.
31.	Stuck K.C. & Wang S.Y. (1996). Establishment and persistence of Baculovirus penaei infections in cultured Pacific white shrimp Penaeus vannamei. J. Invertebr. Pathol., 68, 59-64.
32.	Summers M.D. (1977). Characterization of Shrimp Baculovirus. U.S. Environmental Protection Agency Report, EPA-600/3-77-130, US E.P.A., Gulf Breeze, FL, USA, 35 pp.
33.	Thurman R.B., Lightner D.V., Bell T.A. & Hazanow S. (1990). Unique physicochemical properties of the occluded penaeid shrimp baculoviruses and their use in diagnosis of infections. J. Aquat. Anim. Health, 2, 128-131.
34.	Van Regenmortel M.H.V., Fauquet C.M., Bishop D.H.L., Carstens E.B., Estes M.K., Lemon S.M., Maniloff J., Mayo M.A., McGeoch, D.J., Pringle C.R. & Wickner R.B. (2000). Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses. Academic press, San Diego, USA, 1162 pp.
35.	Wang S.Y., Hong C. & Lotz J.M. (1996). Development of a PCR procedure for the detection of Baculovirus penaei in shrimp. Dis. Aquat. Org., 25, 123-131.


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