From June to October in 1993~1995, cultured Penaeus chinesis and Penaeus japonicus occurred mass mortality at the farm in Western Sea of Korea. The disease was reproduced in healthy shrimp by injection of filtered (at 0.45 micromeger) homogenate of infected shrimp. So we concluded hat it was filterable agents like virus. Clinical symptoms were white spots on the carapace and reddish tail. Histopathological changes were characterized by hypertrophied nuclei at cuticular epidermis lymphoid organ, hematopoietic tissue. In negatively stained preparation, the virion revealed rod-shaped, enveloped, nonoccluded. Cytopathic effect (CPE) were not observed by virus in CHSE-214, RTG-2, EPC, FHM cell lines. Base on the above facts, the reason of mass mortality of penaied shrimp was baculovirus.
Baculoviridae ; Cell Line ; Dental Caries ; Epidermis ; Korea ; Mortality ; Penaeidae* ; Tail ; Virion ; Virus Diseases*

Nucleases is an important enzyme widely used in biotechnology. A codon optimized nuclease gene (SNU) from Northern Shrimps was inserted into pPICZα A vector, and expressed extracellularly in strain SMD1168H. On the basis of multi-copy recombinant strain, we further optimized the expression condition and characterized SNU. SNU was highly expressed and stable after 1% methanol induction for 72 h, yield reached 1.4×10⁵ U/mL. SDS-PAGE electrophoresis demonstrated that this is a N-linked glycoprotein of 50 kDa. It was purified by one step DEAE Sephadex chromatography to the purity of about 15 mg/L with a specific activity of 6.291×10⁶ U/mg. Functional analysis on the nuclease activity indicated that it was stimulated by bivalent iron, such as Ca²⁺, Mn²⁺, Co²⁺ and Mg²⁺, but inhibited by Zn²⁺, Cu²⁺ and high salt. Meanwhile, it was irreversibly inactivated at 70 ℃ for 10 min.
Animals ; Codon ; Electrophoresis, Polyacrylamide Gel ; Endonucleases ; biosynthesis ; Glycoproteins ; Hot Temperature ; Penaeidae ; enzymology ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis

Loop-mediated isothermal amplification (LAMP) assay is a novel method of gene amplification with high specificity, sensitivity and rapidity, which can be applied for disease diagnosis in shrimp aquaculture. The method is performed under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. In the present study, according to the conservative regions of non-structural protein gene NS1, a set of four specific primers were designed, and a rapid detection of IHHNV was established by LAMP assay. The parameters of reaction time and temperature were optimized, and its specificity and sensitivity were assessed. The reactions were carried out at 60 degrees C, 62 degrees C, 63 degrees C, 64 degrees C, 65 degrees C, 66 degrees C, 67 degrees C, 68 degrees C for different time (0 min; 15 min; 30 min; 45 min; 60 min; 75 min). A plasmid pMDIHHNV carrying target sequence of LAMP detection was constructed. Ten-fold serially diluted pMDIHHNV (10(7)-10(0)copies/microL) was used as template for LAMP assay to investigate the detection limit. To determine the specificity, LAMP assays were carried out with DNA templates from other pathogens (White spot syndrome virus; WSSV, Taura Syndrome Virus; TSV, Aeromonas. hydrophila, V. alginolyticus, Vibrio. parahaemolytious, Escherichia. coli). The results showed the optimized LAMP assay for the rapid detection of IHHNV was performed at 65 degrees C for 60 min. The LAMP assay had an unequivocal detection limit of 100 copies/microL, and it was 1,000 times lower than that of PCR. The nucleic acids of other pathogens were not amplified by this LAMP system with the specific primers, which showed a good specificity. The resulting amplicons were detected using visual observation after the addition of SYBR Green I and gel electrophoresis. We investigated the efficacy of UNG (uracil-N-glycosylase) and dUTP in avoiding carry-over contamination in the LAMP assay procedure and explored its effect on the amplification efficiency. Products of LAMP with dUTP adding could be lysed by UNG to avoid LAMP products carry-over contamination effectively. The LAMP assay could be finished within an hour, requiring only a regular laboratory water bath or heat block for reaction and the result could easily be detected using visual observation. Clinically suspected IHHNV-infected shrimp samples were detected by both LAMP and PCR assay, and the result indicated that IHHNV was detected rapidly by LAMP instead of by PCR.
Animals ; DNA Primers ; genetics ; Densovirinae ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; methods ; Penaeidae ; virology ; Viral Proteins ; genetics

Comparative analysis of variable region ORF14/15 genes of white spot syndrome virus (WSSV) genome in Guangxi Penaeus vannamei (P. vannamei) could provide useful information for the evaluation of genetic diversity and genetic evolutionary relationship among WSSV isolates from Guangxi, China and other places. Based on geographical and temporal considerations, 40 WSSV-positive P. vannamei samples were collected during the period between May 2010 and July 2013 from Beihai, Qinzhou, and Fangchenggang, which were the main P. vannamei production areas in Guangxi, and the variable region ORF14/15 genes of the WSSV genome from all infected samples were amplified by PCR and then subjected to cloning and sequence analysis. Pairwise and multiple alignment analysis was then conducted to evaluate the degree of genetic divergence between different strains. The variable region ORF14/15 genes from 25 of 40 WSSV positive samples were successfully cloned and sequenced; among the ORF14/15 genes of 25 WSSV-positive strains, 22 was 619 bp in length and 3 was 620 bp. All the 25 Guangxi strains carried a 5949-bp deletion in the ORF14/15 region relative to TH-96-II, which has the longest nucleotide sequence in this region; the deletion of Guangxi strains occurred in the middle region of ORF14/15 gene, with only 190 bp and 429 bp/ 430 bp at 5' and 3' ends, respectively, which were coincident with WSSV-IN-05-I in deletion length and position. Sixteen of 25 Guangxi strains had completely identical nucleotide sequences in the variable re gion, and the homology between other strains also exceeded 97.9%. There were single nucleotide substi tution, deletion, and insertion in the ORF14/15 region of Guangxi strains compared with other strains in GenBank. In the phylogenetic tree based on WSSV variable region ORF14/15, the Guangxi strains were closely related and formed a separate branch with Indian strain IN-05-I, but far from other strains in GenBank. The ORF14/15 gene of WSSV isolates in cultured P. vannamei in Guangxi has a large deletion in the middle of the variable region, and the Guangxi WSSV strains show no significant spatio-temporal differences; the Guangxi strains are closer in genetics to Indian strain IN-05-I than other strains in GenBank.
Animals ; China ; Cloning, Molecular ; Evolution, Molecular ; Genome, Viral ; genetics ; Genomics ; Penaeidae ; virology ; Phylogeny ; White spot syndrome virus 1 ; genetics

The tropomyosin fraction of shrimp proteins is potentially responsible for allergic reaction in individuals with genetic predisposition to allergy. However, there are no efficient and safe methods to reduce its allergenicity. High intensity ultrasound is known to change the structure of proteins. This study is aimed at assessing high intensity ultrasound's effect on the allergenicity of shrimp allergen. Shrimp and purified shrimp allergen were treated with high intensity ultrasound for 30-180 min. Extracts of treated samples were analyzed by enzyme-linked immunosorbent assay (ELISA) with pool serum of shrimp allergy patients and polyclonal anti-allergen antibodies and by immunoblotting after polyacrylamide gel electrophoresis. Shrimp treated with high intensity ultrasound showed a decrease in allergenicity measured with ELISA. A linear relationship between the immune response induced by treated shrimp allergen and the applied treatment time was observed. The decrease in allergenicity was confirmed by immunoblot assays with shrimp allergic patients serum. Allergenicity of shrimp allergen extracted from treated shrimp was higher than that of purified shrimp allergen with the same treatment time. Gel-filtration HPLC was applied for analysis of shrimp allergen after treatment with high intensity ultrasound. Some fractions were appeared with increasing treatment time. The results suggested that high intensity ultrasound could be used to reduce the allergenicity of shrimp.
Adolescent ; Allergens ; Animals ; Arthropod Proteins ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Food Hypersensitivity ; immunology ; prevention & control ; Humans ; Penaeidae ; immunology ; Proteins ; chemistry ; immunology ; Ultrasonography ; methods

BACKGROUND: Shrimp is one of the major causative crustacean food allergen. An investigation has been reported that tropomyosin belonged to muscle protein is a major allergen within shrimp. But there have been a few investigations on shrimp allergen in Korea. The aim of this study is to evaluate skin reactivity and specific IgE sensitization to Metapenaeus joyneri which is one of the major shrimp in this country, and to identitify IgE binding components and evaluate allergenic relationship with other species. METHODS: We performed skin prick test with M. joyneri extract in 1,738 patients. ELISA was performed for detection of serum specific IgE antibody. To evaluate the cross allergenecity between M. joyneri and other crustaceans (crab, lobster, crayfish), Dermatophagoides pteronyssinus (Dpt), triton shell, abalone and buckwheat. ELISA inhibition tests were performed with each four patient's sera showing high specific IgE antibody. To identify IgE binding components, SDS-PAGE followed by IgE-Immunoblot were applied. RESULTS: 211 patients (12.2%) showed positive responses (A/H >or=2+) on skin prick test. Serum specific IgE antibodies were detected in 61 patients (37.2%) of 164 sensitzed patients. ELISA inhibition test using four patient's sera showed significant inhibitions by M. joyneri. and other crustaceans including lobster, crab and crayfish, partial inhibitions were noted by Dpt, triton shell, buckwheat and abalone. SDS-PAGE and IgE-imunoblot with patients' individual sera sensitized to M. joyneri showed 12 IgE binding components (31, 32, 38-44, 57, 70, 81 kDa) and two (31, 32 kDa) were bound to IgE in more than 50% of sera tested. Five (43, 44, 57, 70 and 81 kDa) were bound to IgE in more than 25% of sera tested. CONCLUSION: Specific IgE was detected in 37.2% of allergy patients sensitized to M. joyneri. Twelve IgE binding components and two (31, 32 kDa) major allergens were indentified. Cross allergenecity was noted with other crustaceans.
Allergens ; Antibodies ; Astacoidea ; Dermatophagoides pteronyssinus ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Fagopyrum ; Humans ; Hypersensitivity ; Immunoglobulin E* ; Korea ; Muscle Proteins ; Neptune ; Penaeidae ; Skin ; Tropomyosin

According to the conservative sequence in the epitaxial variable region of Thailand strain of WSSV published in GenBank,a pair of primers were designed to amplify the variable region genes of 5 local WSSV strains (ZHSH, ZHJ, HN, QD1, QD2) by PCR and then cloned. In accordance with the CN, the results indicated that the number of nucleotide of 5 strains were deleted more than 591bp of the TW and TH strains. The ZHSH and HN strains that deleted 591bp at the 3' end, and 454 bp at the 5' end of variable gene was highly homologous with CN strain about 99.3%. 229bp of ZHJ strain at the 5' end was homologous with CN about 99.3%, and deleted of 816bp at the 3' end. 97bp at the 5' end and 171bp at the 3' end of QD1 and QD2 strains were homologous with CN strain about 99.3%, and about 777bp were absent in the middle. The above data showed that the variable region genes of WSSV had mutated more in China. The variable region gene of QD2 strain was coincidence with that of QD1 after propagating 10 generations in crayfish. The results indicated that the crayfish inoculation did not result in mutation of variable region genes of WSSV.
Animals ; China ; Gene Deletion ; Genes, Viral ; Genetic Variation ; Mutation ; Penaeidae ; virology ; Plasmids ; Polymerase Chain Reaction ; White spot syndrome virus 1 ; genetics

White spot syndrome virus (WSSV) is one of the most important pathogens in shrimp farm throughout the world. Many researches on WSSV have been done, but no efficient approach has been gained to protect and cure the disease. In this study, we constructed a single-chain fragment variable (scFv) antibody library displayed on phage using spleen cells from mice immunized with denatured WSSV. After several rounds of panning respectively against purified intact WSSV virions and purified VP28 expressed in Escherichia coli, five novel scFv antibodies specifically against WSSV were selected, one of which, clone P75E8, recognized a linear epitope. The location in virions of the epitopes recognized by the five scFv clones was determined by immunoelectron microscopy. This study provides a new way to obtain more different antibodies specifically binding to WSSV, and especially provides a new strategy to obtain scFvs against linear epitopes.
Amino Acid Sequence ; Animals ; Antibodies, Viral ; immunology ; Antibody Specificity ; immunology ; Antigens, Viral ; immunology ; Epitopes ; immunology ; Immunoglobulin Fragments ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Mice ; Molecular Sequence Data ; Penaeidae ; virology ; Peptide Library ; White spot syndrome virus 1 ; immunology

Prawn white spot syndrome is caused by the pathogen prawn white spot syndrome virus (WSSV). VP19 is a vesicle membrane protein of WSSV. HyNPV (Hybrid of AcNPV and BmNPV) constructed by the recombination of BmNPV and AcNPV is a new hybrid virus having both of their advantages. The recombinant transfer vector pBlueBicHisC-vp19 and recombinant baculovirus HyNPV-VP19 were constructed on the basis of the successful cloning of VP19. Newly-molted silkworms Bombyx mori of fifth instar were inoculated by the recombinant virus. SDS-PAGE and Western blotting analysis showed a specific band, about 21kD, which was consistent with the expectation suggesting that the WSSV-VP19 gene was successfully expressed in silkworm bodies.
Animals ; Baculoviridae ; genetics ; metabolism ; Bombyx ; genetics ; metabolism ; virology ; Genetic Vectors ; Penaeidae ; virology ; Recombinant Fusion Proteins ; genetics ; Viral Envelope Proteins ; genetics ; metabolism ; Virus Replication ; White spot syndrome virus 1 ; genetics

BACKGROUND: Black tiger shrimp Penaeus monodon is one of the common causes of shellfish allergy that is increasing worldwide. One of the important problems in the management of shellfish allergy is the lack of accurate diagnostic assay because the biological and immunological properties of allergens in black tiger shrimp have not been well characterized. This study aims to detect proteins with the ability to bind and cross-link immunoglobulin E (IgE) from black tiger shrimp by enzyme-linked immunosorbent assay (ELISA), Western blot, and a humanized rat basophilic leukemia reporter cell line RS-ATL8. METHODS: Sera from shrimp allergic subjects were subjected to ELISA and Western blots using raw or cooked shrimp extract as antigens. Pooled sera were used to sensitize the RS-ATL8 reporter cell line and cells were activated by shrimp extract. Eluted protein extracts separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were tested on the RS-ATL8 cell line and subjected to mass spectrometry to identify potential candidate allergens. RESULTS: Allergic sera reacted stronger to raw shrimp extract than cooked shrimp extract (P=0.009). Western blot demonstrated that major IgE reactivity protein bands were at 32–39 kDa and 91–230 kDa in both raw and cooked shrimp extracts. The eluted protein bands at the molecular weight of 38 and 115 kDa from raw shrimp extract induced IgE cross-linking as assayed by the RS-ATL8 cell line. These protein bands were subjected to mass spectrometry for analysis. Ubiquitin-activating enzyme and crustacyanin were identified as potential candidate novel shrimp allergens. CONCLUSIONS: The RS-ATL8 reporter cell line can be used to identify potential new shrimp allergens that can functionally cross-link IgE and induce mast cell degranulation.
Allergens ; Animals ; Basophils ; Blotting, Western ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin E ; Immunoglobulins ; Leukemia ; Mass Spectrometry ; Mast Cells ; Molecular Weight ; Penaeidae ; Rats ; Shellfish Hypersensitivity ; Sodium Dodecyl Sulfate ; Tigers ; Ubiquitin-Activating Enzymes