As one of the most important aquatic fish, Micropterus salmoides suffers lethal and epidemic disease caused by rhabdovirus at the juvenile stage. In this study, a new strain of M. salmoides rhabdovirus (MSRV) was isolated from Yuhang, Zhejiang Province, China, and named MSRV-YH01. The virus infected the grass carp ovary (GCO) cell line and displayed virion particles with atypical bullet shape, 300-500 nm in length and 100-200 nm in diameter under transmission electron microscopy. The complete genome sequence of this isolate was determined to include 11 526 nucleotides and to encode five classical structural proteins. The construction of the phylogenetic tree indicated that this new isolate is clustered into the Vesiculovirus genus and most closely related to the Siniperca chuatsi rhabdovirus. To explore the potential for a vaccine against MSRV, a glycoprotein (1-458 amino acid residues) of MSRV-YH01 was successfully amplified and cloned into the plasmid pFastBac1. The high-purity recombinant bacmid-glycoprotein was obtained from DH10Bac through screening and identification. Based on polymerase chain reaction (PCR), western blot, and immunofluorescence assay, recombinant virus, including the MSRV-YH01 glycoprotein gene, was produced by transfection of SF9 cells using the pFastBac1-gE2, and then repeatedly amplified to express the glycoprotein protein. We anticipate that this recombinant bacmid system could be used to challenge the silkworm and develop a corresponding oral vaccine for fish.
Animals ; Baculoviridae/metabolism* ; Bass/metabolism* ; Carps/virology* ; Cell Line ; Female ; Genetic Techniques ; Genome, Viral ; Glycoproteins/biosynthesis* ; Insecta ; Ovary/virology* ; Phylogeny ; Plasmids/metabolism* ; Recombinant Proteins/biosynthesis* ; Rhabdoviridae/metabolism*

The antigenicity of Photobacterium damselae (Ph. d.)subsp. piscicida, cultured in four different growth media[tryptone soya broth (TSB), glucose-rich medium (GRM),iron-depleted TSB (TSB+IR-), and iron-depleted GRM(GRM+IR-)] was compared by enzyme-linked immuno-sorbent assay (ELISA) and Western blot analysis usingsera obtained from sea bass (Dicentrarchus labrax) raisedagainst live or heat-killed Ph. d. subsp. piscicida. Theantigenic expression of Ph. d. subsp. piscicida was found todiffer depending on the culture medium used. A significantlyhigher antibody response was obtained with iron-depletedbacteria by ELISA compared with non-iron depletedbacteria obtained from the sera of sea bass raised againstlive Ph. d. subsp. piscicida. The sera from sea bass raisedagainst live bacteria showed a band at 22kDa in bacteriacultured in TSB+IR- or GRM+IR- when bacteria thathad been freshly isolated from fish were used for thescreening, while bands at 24 and 47kDa were observedwith bacteria cultured in TSB or GRM. When bacteriawere passaged several times on tryptic soya agar prior toculturing in the four different media, only bands at 24 and47kDa were recognized, regardless of the medium used toculture the bacteria. It would appear that the molecularweight of Ph. d. subsp. piscicida antigens change in thepresence of iron restriction, and sera from sea bassinfected with live bacteria are able to detect epitopes onthe antigens after this shift in molecular weight.
Animals ; Antibodies, Bacterial/blood ; Antigens, Bacterial/immunology/*metabolism ; Bass/blood/*immunology ; Blotting, Western/veterinary ; Cell Count/methods ; Culture Media ; Enzyme-Linked Immunosorbent Assay/veterinary ; Fish Diseases/immunology/*microbiology ; Molecular Weight ; Pasteurella Infections/immunology/microbiology/*veterinary ; Photobacterium/*immunology