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

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

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

To compare the activity of different promoter in baculovirus-insect system, a series of recombinant baculoviruses were generated harboring the E-GFP reporter gene under the control of one of 5 promoters, including the ie1 promoter of shrimp white spot syndrome virus (WSSV), the truncated ie1 (mie1) promoter, the ETL promoter of the baculovirus, the elongated ETL (mETL) promoter, and the polyhedron promoter (P(PH)) of the baculovirus. The expression efficiency of the E-GFP reporter gene in the recombinant baculovirus-infected Sf9 cells was determined by flow cytometry. The results showed that both ie1 and mETL promoters had a strong promoter activity at early phase, while P(PH) showed a strong promoter activity at late phase. The ie1 promoter suggested the strongest promoter activity. The homologous region 1 (hr1) was also found to enhance the ETL promoter activity.
Animals ; Baculoviridae ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Insecta ; genetics ; metabolism ; Penaeidae ; virology ; Promoter Regions, Genetic ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; 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

White spot syndrome virus (WSSV) is a major pathogen in aquaculture penaeid shrimp, which caused catastrophic economic losses in the worldwide. No adequate treatments against WSSV are available. In order to study infection mechanism of WSSV, a phage display scFv cDNA library against WSSV was constructed and a neutralizing antibody of scFv P1D3 was selected in our lab previously. In this study, scFv P1D3 was expressed successfully in yeast. Firstly, the original expression vector of P1D3, M13 phagmid, was used as a template to design primers with restriction sites of SnaB I and EcoR I . Then the gene of P1D3 was amplified by PCR. After digested by SnaB I and EcoR I , the fragment of scFv P1D3 with E-tag was inserted into yeast and E. coli shuttle plasmid pPIC9k. The recombinant plasmid pPIC9k-scFv P1D3-Etag was linearized with Bgl II and then transformed into Pichia pastoris GS115 by electroporation. Positive clones were selected and verified by PCR and DNA sequencing. The scFv PID3 was induced to express in yeast by methanol. The results of ELISA demonstrate that scFv P1D3 expressed in yeast still has high specificity to bind on WSSV and the binding activity is higher than that expressed in E. coli TG1. After several optimizing experiments, the results show that the expression amount of scFv P1D3 can reach to 302 mg/L in yeast culture supernatant. This experiment has offered a new source of antibody for the researches on passive immunology for shrimp.
Animals ; Antibody Specificity ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; drug effects ; Methanol ; pharmacology ; Penaeidae ; virology ; Pichia ; drug effects ; genetics ; Single-Chain Antibodies ; analysis ; biosynthesis ; genetics ; immunology ; Temperature ; White spot syndrome virus 1 ; immunology