Development and characterization of monoclonal antibodies to spring viraemia of carp virus

Five monoclonal antibodies (mAbs) against spring viraemia of carp (SVCV0504, isolated from common carp in China) were produced from mice immunized with purified virus preparations. The virion of SVCV contains five structural proteins, representing the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (Q. Western blotting analysis revealed that three mAbs (1145, IE10, and 11-17) recognized specifically to a single protein of 47 kDa (N), the mAb 3G4 reacted with, two SVCV0504 proteins of 69 kDa (G) and 47 kDa (N), while the mAb 1A9 reacted with three SVCV0504 proteins of 69 kDa (G), 50 kDa (P), and 47 kDa (N). By indirect ELISA, two mAbs (1H5 and 11-17) showed cross-reactivity with pike fry rhabdovirus (PFRV), but no cross-reactions with the Siniperca chuatsi rhabdovirus (SCRV), Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PoRV) were demonstrated with the five mAbs. Indirect immunofluorescence showed intense fluorescence in the cytoplasm of the SVCV0504-infected epithelioma papulosum cyprini (EPC) cells in areas corresponding to the location of granular structures. The sucrose gradient-purified SVCV0504 particles could be detected successfully by these mAbs using immunodot blotting. mAb 1A9 could completely neutralize 100 TCID50 (50% tissue culture infective dose) of SVCV0504 at a dilution of 1:8. This is the first report of development of the neutralizing mAbs against SVCV. The mAb 1A9 was analyzed further and could be used to successfully detect viral antigens in the infected-EPC cell cultures or in cryosections from experimentally infected crucian carp (Carassius auratus) by immunohistochemistry assay. Furthermore, a flow cytometry procedure for the detection and quantification of cytoplasmic SVCV0504 in cell cultures was developed with mAb 1A9. At 28 h after inoculation with the virus (0.01 PFU/cell), 10.12% of infected cells could be distinguished from the uninfected cells. These mAbs will be useful in diagnostic test development and pathogenesis studies for fish rhabdovirus. (c) 2008 Elsevier B.V. All rights reserved.

Controlling factors of spring-summer phytoplankton succession in Lake Taihu (Meiliang Bay, China)

The spring-summer successions of phytoplankton and crustacean zooplankton were examined weekly in Meiliang Bay of the subtropical Lake Taihu in 2004 and 2005. During the study period, the ecosystem of Meiliang Bay was characterized by (i) clearly declined nitrogen compounds (nitrate, TN, and ammonium) and slowly increased phosphorus compounds (TP and SRP), (ii) increased total phytoplankton density and rapid replacement of chlorophyta (mainly Ulothrix) by cyanobacteria (mainly Microcystis), and (iii) rapid replacement of large-sized crustaceans (Daphnia and Moina) by small-sized ones (Bosmina, Limnoithona, and Ceriodaphnia). Results from the CCA and correlation analysis indicate that the spring-summer phytoplankton succession was primarily controlled by abiotic factors. Cyanobacteria were mainly promoted by increased temperature and decreased concentrations of nitrogen compounds. The pure contribution of crustacean was low for the variation of phytoplankton suggesting a weak top-down control by crustacean zooplankton in the subtropical Lake Taihu.

Characterization of complete genome sequence of the spring viremia of carp virus isolated from common carp (Cyprinus carpio) in China

The complete genome of spring viraemia of carp virus (SVCV) strain A-1 isolated from cultured common carp (Cyprinus carpio) in China was sequenced and characterized. Reverse transcription-polymerase chain reaction (RT-PCR) derived clones were constructed and the DNA was sequenced. It showed that the entire genome of SVCV A-1 consists of 11,100 nucleotide base pairs, the predicted size of the viral RNA of rhabdoviruses. However, the additional insertions in bp 4633-4676 and bp 4684-4724 of SVCV A-1 were different from the other two published SVCV complete genomes. Five open reading frames (ORFs) of SVCV A-1 were identified and further confirmed by RT-PCR and DNA sequencing of their respective RT-PCR products. The 5 structural proteins encoded by the viral RNA were ordered 3'-N-P-M-G-L-5'. This is the first report of a complete genome sequence of SVCV isolated from cultured carp in China. Phylogenetic analysis indicates that SVCV A-1 is closely related to the members of the genus Vesiculovirus, family Rhabdoviridae.

Spatial analysis for spring bloom and nutrient limitation in Xiangxi bay of three Gorges Reservoir

The spatial and temporal dynamics of physical variables, inorganic nutrients and phytoplankton chlorophyll a were investigated in Xiangxi Bay from 23 Feb. to 28 Apr. every six days, including one daily sampling site and one bidaily sampling site. The concentrations of nutrient variables showed ranges of 0.02-3.20 mg/L for dissolved silicate (Si); 0.06-2.40 mg/L for DIN (NH4N + NO2N + NO3N); 0.03-0.56 mg/L for PO4P and 0.22-193.37 mu g/L for chlorophyll a, respectively. The concentration of chlorophyll a and inorganic nutrients were interpolated using GIS techniques. The results indicated that the spring bloom was occurred twice in space during the whole monitoring period (The first one: 26 Feb.-23 Mar.; the second one: 23 Mar.-28 Apr.). The concentration of DIN was always high in the mouth of Xiangxi Bay, and PO4P was high in the upstream of Xiangxi Bay during the whole bloom period. Si seems no obvious difference in space in the beginning of the spring bloom, but showed high heterogeneity in space and time with the development of spring bloom. By comparing the interpolated maps of chlorophyll a and inorganic variables, obvious consumptions of Si and DIN were found when the bloom status was serious. However, no obvious depletion of PO4P was found. Spatial regression analysis could explained most variation of Chl-a except at the begin of the first and second bloom. The result indicated that Si was the factor limiting Chl-a in space before achieved the max area of hypertrophic in the first and second bloom period. When Si was obviously exhausted, DIN became the factor limiting the Chl-a in space. Daily and bidaily monitoring of Site A and B, representing for high DIN: PO4P ratio and low DIN:PO4P ratio, indicated that the concentration of Si was decreased with times at both site A and B, and the dramatically drop of DIN was found in the end monitoring at site B. Multiple stepwise regression analysis indicated that Si was the most important factor affect the development of spring bloom both at site A and B in time series.

Daily dynamics of nutrients and chlorophyll a during a spring phytoplankton bloom in Xiangxi Bay of the Three Gorges Reservoir

We studied the daily dynamics of nutrients (total phosphorus [TP], total nitrogen [TN], and dissolved silicate [SiO2]) and chlorophyll a (chl a) during a spring bloom in Xiangxi Bay of the Three Gorges Reservoir in year 2005. According to the daily dynamics of chl a, the bloom occurred in two stages (23 February-25 March and 26 March-28 April). The concentration of SiO2 decreased at different layers of the water column with the development of the bloom. However, the decrease of SiO2 in the layers with high concentration of chl a was more dramatic than in the layers with low concentration of chl a. The concentration of TP was lowest value a few days after the peak of chl a during the first bloom period, and the lowest value of TN was found a few days after the peak of chl a during the second bloom period. Correlative analyses indicated that SiO2 and TP were limiting factors in the first bloom period, and SiO2 and TN were limiting factors in the second bloom period.

Detection of extracellular phosphatases in natural spring phytoplankton of a shallow eutrophic lake (Donghu, China)

The species-specific production of extracellular phosphatases in phytoplankton of a subtropical polymictic take was investigated from March to May 2004. Phosphatase activity was detected directly at the site of enzyme action using the enzyme-labelled fluorescence (ELF) technique. Size fractionation of bulk phosphatase activity (PA), concentrations of soluble reactive phosphorus (SRP), chlorophyll a, and phytoplankton composition were determined in parallel. Phosphatase-positive cells were present in every phytoplankton sample; labelled cells were detected in 33 algal taxa, including many chlorophytes, dinoflagellates and some diatoms, but never among cyanobacteria. We recorded an unusual dinoflagellate bloom (Peridiniopsis sp.), of which similar to 25% of the cells were phosphatase-positive. Several populations were partly phosphatase-positive whenever present, while some other species never showed any activity. The production of extracellular phosphatases was not primarily regulated by ambient P concentrations; algae produced these enzymes even if SRP concentrations were high. Moreover, heterotrophic nanoflagellates most probably contributed to the pool of particle-bound PA in some samples.