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IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C-terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0. [ABSTRACT FROM AUTHOR]

Background: White spot syndrome virus (WSSV) is an important pathogen of the penaeid shrimp with high mortalities. In previous reports, Orf427 of WSSV is characterized as one of the three major latency-associated genes of WSSV. Here, we were interested to analyze the promoter of orf427 and its expression during viral pathogenesis. Results: in situ hybridization revealed that orf427 was transcribed in all the infected tissues during viral lytic infection and the translational product can be detected from the infected shrimp. A time-course RT-PCR analysis indicated that transcriptional products of orf427 could only be detected after 6 h post virus inoculation. Furthermore, a baculovirus-mediated promoter analysis indicated that the promoter of orf427 failed to express the EGFP reporter gene in both insect SF9 cells and primary shrimp cells. Conclusion: Our data suggested that latency-related orf427 might not play an important role in activating virus replication from latent phase due to its late transcription during the lytic infection. [ABSTRACT FROM AUTHOR]

Infection of Spodoptera littoralis SL2 cells with the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) results in apoptosis and low yields of viral progeny, in contrast to infection with S. littoralis nucleopolyhedrovirus (SINPV). By cotransfecting SL2 cells with AcMNPV genomic DNA and a cosmid library representing the complete SINPV genome, we were able to rescue AcMNPV replication and to isolate recombinant virus vAcSL2, which replicated efficiently in SL2 cells. Moreover, vAcSL2 showed enhanced infectivity for S. littoralis larvae compared to AcMNPV. The genome of vAcSL2 carried a 519-bp insert fragment that increased the distance between the TATA element and the transcriptional initiation site (CAGT) of immediate-early gene ieO. This finding correlated with low steady-state levels of IE0 and higher steady-state levels of IE1 (the product of the ie1 gene, a major AcMNPV transactivator, and a multifunctional protein) than of IE0. Mutagenesis of the ieO promoter locus by insertion of the chloramphenical acetyltransferase (cat) gene yielded a new recombinant AcMNPV with replication properties identical to those of vAcSL2. Thus, the analysis indicated that increasing the steady-state levels of lEI relative to IE0 should enable AcMNPV replication in SL2 cells. This suggestion was confirmed by constructing a recombinant AcMNPV bearing an extra copy of the ie1 gene under the control of the Drosophila hsp70 promoter. These results suggest that IE0 plays a role in the regulation of AcMNPV infection and show, for the first time, that significant improvement in the ability of AcMNPV to replicate in a poorly permissive cell line and organism can be achieved by increasing the expression of the main multiple functional protein, IE1. [ABSTRACT FROM AUTHOR]

Cyprinid herpesvirus 2 (CyHV-2), a member of the Alloherpesviridae family belonging to the genus Cyprinivirus, was initially isolated from goldfish (Carassius auratus) and has been recently emerging as a virulent pathogen for cultured prussian carp (Carassius auratus gibelio) world-wide. In this study, a novel and effective method for concentration and quantification of live CyHV-2 virions from water was successfully established through coupling the iron flocculation with real time qPCR assay. Then, the shedding intensity of CyHV-2 in fish-tank water from artificially-challenged goldfish (25 fish/20 L) was monitored continuously for 7 days on a daily basis through quantitating viral genomic copy numbers by qPCR, and the maximum shedding level was determined to be 105 copies/L. Horizontal transmission research system was established by inoculating healthy goldfish in water spiked with serial dilution of CyHV-2 virions ranging from 107 to 10³ copies/L. Our results indicated that water-borne CyHV-2 efficiently caused the infection of tested goldfish even in a concentration of 10³ copies/L, and the overall transmission efficacy was not linearly correlated with the level of input virus in the fish tank. Commercial disinfectant Composite Sodium Chlorite Powder (CSCP) has been widely applied in aquaculture to control microbial infection through direct spill in the water, and its effect in inactivating the CyHV-2 infectivity remains unknown. We further determined that the EC50 of CSCP against 3.89 TCID50/mL CyHV-2 was close to 15.625 µg/mL in vitro, and application of CSCP in a level as high as 60 µg/mL (the safety concentration of CSCP for goldfish) couldn't protect goldfish from CyHV-2 challenge through immersion. Thus, the disinfectant CSCP was regarded as none-effective for blocking CyHV-2 transmission in water during epidemic. Overall, our data provided quantitative data to demonstrate the shedding intensity of CyHV-2 in water, and CSCP was shown to be not effective in blocking water-borne horizontal transmission of CyHV-2 in goldfish. The virus-concentration protocol and virus-inhibition assay established here also paved the way for evaluating more commercial disinfectants in their effects in blocking water-borne horizontal transmission of CyHV-2. [ABSTRACT FROM AUTHOR]

Crucian carp (Carassius auratus gibelio), an extensively cultivated freshwater fish, was one of the model species for the study of fish immunology. Polyclonal antibodies were advantageous molecular tools for studying teleost immune system. Specifically, polyclonal antibodies reacting with immunoglobulins (Ig) were used successfully in studies of the teleost fishes. In the present study, we produced polyclonal antibody against CH2 domains of crucian carp IgM, and measured the in vivo dynamics of IgM mRNA caused by CyHV-2 infection. The recombinant protein IgM with relative molecular weight about 53 KD was correctly expressed in prokaryotic cells. The specificity of the polyclonal antibody was evaluated by Western blotting and results revealed that the antibody not only specifically recognized crucian carp serum but also cross-reacted with grass carp serum. Quantitative RT-PCR analysis demonstrated the expression of IgM mRNA changed significantly after CyHV-2 infection. The expression of IgM in the kidney increased and reached a maximum at 6 h post-infection (hpi), while dropped to a low level at 5 days post-infection (dpi). In conclusion, the expression of IgM was significantly upregulated in the kidney of crucian carp infected with CyHV-2, indicating that IgM played a potential role in systemic immunity against viral infection. Polyclonal antibody against crucian carp IgM had certain clinical relevance, which might provide insight into the early stage of virus infection and prevention of the disease. [ABSTRACT FROM AUTHOR]

Ulcerative disease causes significant economic losses in largemouth bass Micropterus salmoides production. Yet information reported on Bacillus mycoides as a pathogen for largemouth bass is scarce. In this study, a virulent strain, temporarily named LYS1, was isolated from diseased M. salmoides suffering from ulcerative disease, identified phenotypically and molecularly as B. mycoides. A phylogenetic tree was constructed to examine isolate LYS1 and compare it to other known isolates. In addition, isolate LYS1 appears to be susceptible to aminoglycosides and quinolones drugs for veterinary use in aquaculture as seen when screened against a range of common antibiotics. To the best of our knowledge, this is the first report of B. mycoides as a pathogen of ulcerative disease in farmed largemouth bass. [ABSTRACT FROM AUTHOR]

Presents a correction to an article published in the September 2005 issue of "Journal of Virology."

Activating protein-1 (AP-1) composed of Jun and Fos family proteins, which are frequently activated by pathogens to induce cellular inflammation and immune responses. In this study, by using RNA sequencing (RNA-seq) technology, we found that FosB, Fosl2, and JunD expression was co-induced 12 and 20 h in grass carp (Ctenopharyngodon idella) kidney cell line (CIK cells) after grass carp reovirus (GCRV) infection. Full-length cDNA of the three genes were cloned and phylogenetic analysis indicated that the corresponding proteins shared high homology with those in higher vertebrates. The tissue distribution analysis of the three genes showed they were ubiquitously expressed in various tissues of healthy grass carp. In infection assay, FosB, Fosl2, and JunD were upregulated and co-expressed 8 h after GCRV infection in CIK cells. Protein interaction and co-expression network analysis revealed that FosB, Fosl2, and JunD showed similar functions and were co-expressed in mammals and fish. Taken together, these results indicated that the co-expression of the AP-1 family proteins in grass carp uniformly responds to GCRV infection. These findings provided insights into the further study of AP-1 transcription factors function during grass carp reovirus infection. [ABSTRACT FROM AUTHOR]

Waterlogging in the early stage of cotton will reduce the number of bolls and do harm to yield. Early detection of waterlogging will help farmers to adjust cotton management and save the loss. To evaluate the application of deep learning for the detection of early waterlogging, this study applied a convolutional neural network (CNN) to classify different durations of waterlogging stress (0, 2, 4, 6, 8, 10 d) based on hyperspectral images (HSIs) of cotton leaves. An experiment was designed to simulate the situation of cotton under waterlogging stress and collect HSIs of visible and near-infrared (VNIR 450-950 nm) spectra with 126 bands 66 d after cotton sowing (66 DAS). It was found the spectral curve reflectance of waterlogging cotton was higher than that of non-waterlogging cotton. Especially near 550 nm and 750 nm, and the spectral curve increased with durations of waterlogging stress and there were 'blue shift' phenomena for the position of the red edge of the spectra. The first principal components of HSIs after band randomly discarding and principal component analysis (PCA) were used to build a dataset. GoogLeNet Inception-v3 (GLNI-v3) and VGG-16 models were selected to detect cotton waterlogging stress with the dataset. The results showed that the average time for a round training for GLNI-v3 was 13.337 s, with a classification accuracy of 96.95% and a loss value of 0.09431. The average time for a round training for VGG-16 was 21.470 s, with a classification accuracy of 97.00% and a loss value of 0.17912. Though these two models had similar classification accuracy and loss value, GLNI-v3 achieved a high accuracy with fewer training iterations. The durations of waterlogging stress of cotton in a short-term can be detected by HSIs of cotton leaves and CNN models are suitable for the classification of HSIs, and this method can provide support for cotton yield estimation and loss assessment after waterlogging. [ABSTRACT FROM AUTHOR]

The drying shrinkage caused by rapid water loss will pose a great threat to the safety of concrete structures, especially for those serviced in an extremely dry environment. However, the majority of reported research primarily focuses on drying shrinkage behavior of concrete under relative humidity (RH) ranging from 50% to 100%, and the corresponding drying shrinkage prediction models have poor applicability, leading to an unclear understanding of it under ultra-low RH (RH<40%). In this study, a numerical model, finite element model with cohesive elements, was proposed to better predict the drying shrinkage behavior of concrete, even under the RH of 20%. Initially, geometric models of concrete with varying coarse aggregate size ranges and volume fractions were established. Subsequently, based on the relationship between RH and humidity diffusion coefficient of concrete with different water-cement ratio (w/c), a drying shrinkage model was proposed to examine the corresponding shrinkage strain of concrete even under ultra-low RH conditions, and its validity was verified through comparison with reported experimental research. Utilizing the developed model, the effects of size range and volume fraction of coarse aggregate, and the w/c, on the drying shrinkage of concrete across a wide range of RH levels were systematically investigated. The results indicated that increasing the aggregate size range can only slightly inhibit drying shrinkage, while there is a marked inhibition effect on it (decrease by about 45% at most) for increasing the aggregate vol% or reducing w/c, even under ultra-low RH of 20%. Finally, grey relation analysis was employed to assess the inhibition weight of each parameter on the drying shrinkage, following this sequence in order: RH > w/c > coarse aggregate vol% > coarse aggregate range size. We also envision that the proposed model could provide feasible guidelines for the inhibition of concrete in ultra-dry regions. • A numerical drying shrinkage prediction model was proposed. • Effect of aggregate, w/c and RH on drying shrinkage strain was studied. • The inhibition weight of each parameter on the drying shrinkage was explored. [ABSTRACT FROM AUTHOR]

Enteritis has caused significant economic losses in farmed channel catfish Ictalurus punctatus. Yet information is limited on Enterobacter aerogenes as a potential causal agent for enteritis in channel catfish. In this study, a virulent strain, temporarily named HT2, was isolated from diseased channel catfish suffering from enteritis, identified phenotypically and molecularly as E. aerogenes. A phylogenetic tree was constructed to examine isolate HT2 and compare it to other known isolates. In addition, isolate HT2 is apparently susceptible to aminoglycosides and quinolones, drugs for veterinary use in aquaculture as seen when screened against a range of common antibiotics. To the best of our knowledge, this is the first report of E. aerogenes as a pathogen causing enteritis in farmed channel catfish. [ABSTRACT FROM AUTHOR]

Bacteriosis has caused significant economic losses in farmed blunt snout bream Megalobrama amblycephala. Only scarce information is available on Acinetobacter johnsonii as a possible causal agent in M. amblycephala. In this study, a virulent strain temporarily named BY3, was isolated from diseased bream, identified phenotypically and molecularly as A. johnsonii. Phylogenetic trees of 16S rRNA and gyrB gene sequences were constructed to examine the taxonomic position of isolate BY3 and compare it to other known isolates. When screened against a range of common veterinary antibiotics, isolate BY3 has not shown resistance to florfenicol, neomycin, and sulfisoxazole antibiotics in aquaculture. To the best of our knowledge, this is the first report of A. johnsonii infection in farmed blunt snout bream. [ABSTRACT FROM AUTHOR]

Background: Grass carp reovirus (GCRV) is responsible for viral hemorrhagic disease in cultured grass carp (Ctenopharyngon idellus). GCRV is a non-enveloped, double-stranded RNA virus in the genus Aquareovirus,o f the family Reoviridae, which encodes seven structural proteins (VP1-VP7) and five nonstructural proteins (NS80, NS38, NS31, NS26, and NS16). To date, the mechanism of GCRV entry into CIK Ctenopharyngon idellus kidney (CIK) cells remains poorly understood. Results: Here, we present a study of the GCRV internalization mechanism in CIK cells. Our results indicated that GCRV infection was inhibited by chlorpromazine, the specific inhibitor for clathrin-mediated endocytosis. Colocalization of GCRV virions with endogenous clathrin was observed during early infection by confocal microscopy. Moreover, GCRV infection of CIK cells depended on acidification of the endosome. This was indicated by significant inhibition of viral infection following prophylactic treatment with the lysosomotropic drugs chloroquine or ammonium chloride. In addition, the disturbance of dynamin activity blocked GCRV entry, which confirmed the dynamin-dependent nature of clathrin-mediated endocytosis. Conclusion: Our findings suggest that GCRV might enter CIK cells via clathrin-mediated endocytosis in a pH-dependent manner. Additionally, dynamin is critical for efficient viral entry. [ABSTRACT FROM AUTHOR]

Whiteleg shrimp Penaeus vannamei is an important commercial shrimp species cultivated in China and many other countries worldwide. Bacteriosis is a major economic problem that inhibits the farming of this species in fresh water. White feces syndrome is an emerging epidemic in freshwater cultured P. vannamei and has caused significant economic damage. Only scarce information is available on Vibrio cholerae as a possible causal agent for this disease. In this study, a virulent strain BB31 was isolated from diseased P. vannamei suffering from white feces syndrome, and identified as a V. cholerae isolate through phylogenetic analysis and phenotypic characteristics. A phylogenetic tree was constructed to examine the relationship of isolate BB31 to other V. cholerae isolates. Three genes encoding hemolysin, outer membrane protein, and cholera toxin transcriptional activator were present in the BB31 isolate confirming its potential pathogenicity. In addition, isolate BB31 is known to have developed resistance to penicillin, sulfonamides and cephalosporin antibiotics. This was demonstrated when screened against a range of common antibiotics for aquaculture and veterinary use. To the best of our knowledge, this is the first report of white feces syndrome caused by V. cholerae in freshwater farmed P. vannamei. [ABSTRACT FROM AUTHOR]

Bacteriosis has become a major global economic problem in freshwater farmed whiteleg shrimp Penaeus vannamei. However, limited information is available on the incidence of Aeromonas schubertii infections in freshwater cultured P. vannamei. Red body disease, an epidemic frequently associated with P. vannamei freshwater farming, occurred in a P. vannamei farm in Shanghai China in June 2013. A pathogenic strain of A. schubertii (isolate HS1) was isolated from diseased freshwater cultured P. vannamei suffering from red body disease, and identified through phylogenetic analysis and phenotypic characteristics. A phylogenetic tree was constructed to examine the relatedness of isolate HS1 with other A. schubertii isolates. In addition, isolate HS1 showed no signs developing antibiotic resistance when screened against a range of common antibiotics used in aquaculture. To the best of our knowledge, this is the first report of A. schubertii infection in freshwater farmed P. vannamei. [ABSTRACT FROM AUTHOR]

Background: Antibiotic resistance has become a serious global problem and is steadily increasing worldwide in almost every bacterial species treated with antibiotics. In aquaculture, the therapeutic options for the treatment of A. hydrophila infection were only limited to several antibiotics, which contributed for the fast-speed emergence of drug tolerance. Accordingly, the aim of this study was to establish a medication regimen to prevent drug resistant bacteria. To determine a rational therapeutic guideline, integrated pharmacodynamics and pharmacokinetics parameters were based to predict dose and dosage interval of enrofloxacin in grass carp Ctenopharyngodon idella infected by a field-isolated A. hydrophila strain. Results: The pathogenic A. hydrophila strain (AH10) in grass carp was identified and found to be sensitive to enrofloxacin. The mutant selection window (MSW) of enrofloxacin on isolate AH10 was determined to be 0.5 - 3 μg/mL based on the mutant prevention concentration (MPC) and minimum inhibitory concentration (MIC) value. By using high-performance liquid chromatography (HPLC) system, the Pharmacokinetic (PK) parameters of enrofloxacin and its metabolite ciprofloxacin in grass carp were monitored after a single oral gavage of 10, 20, 30 μg enrofloxacin per g body weight. Dosing of 30 μg/g resulted in serum maximum concentration (Cmax) of 7.151 μg/mL, and concentration in serum was above MPC till 24 h post the single dose. Once-daily dosing of 30 μg/g was determined to be the rational choice for controlling AH10 infection and preventing mutant selection in grass carp. Data of mean residue time (MRT) and body clearance (CLz) indicated that both enrofloxacin and its metabolite ciprofloxacin present similar eliminating rate and pattern in serum, muscle and liver. A withdraw time of more than 32 d was suggested based on the drug eliminating rate and pharmacokinetic model described by a polyexponential equation. Conclusions: Based on integrated PK/PD parameters (AUC/MIC, Cmax/MIC, and T>MPC), the results of this study established a principle, for the first time, on drawing accurate dosing guideline for pharmacotherapy against A. hydrophila strain (AH10) for prevention of drug-resistant mutants. Our approach in combining PK data with PD parameters (including MPC and MSW) was the new effort in aquaculture to face the challenge of drug resistance by drawing a specific dosage guideline of antibiotics. [ABSTRACT FROM AUTHOR]

In the present study, a pathogenic strain, JL, was isolated from Pengze crucian carp (Carassius auratus) eggs suffering from saprolegniosis. It was initially determined as Saprolegnia sp. strain JL. Saprolegnia species have been implicated for significant fungal contamination, involving both living and dead fish and their eggs. A phylogenetic tree was constructed using the maximum parsimony method. The tree shows that the JL strain was closely related to Saprolegnia parasitica isolate SAP171, isolated from Salmo trutta suffering from saprolegniasis in Laukaa, Finland. The minimum inhibitory concentration (MIC) of 20 Chinese herbs was screened. Pseudolarix kaempferi Gord. (Pinaceae) was the most effective in inhibiting growth of the bacteria and was chosen for further trial. Significant protective efficacy of 52.63% and 73.68% was obtained against the JL strain in Pengze crucian carp eggs at P. kaempferi concentrations of 12.5 mg/ml and 25.0 mg/ml, respectively. [ABSTRACT FROM AUTHOR]

Recent studies have revealed that the use of probiotics is an alternative to control marine aeromonas. However, few probiotics are available against Aeromonas hydrophila infections in eels. In the present study, a potential antagonistic strain G1 against the eel-pathogenic A. hydrophila was isolated from sediment underlying brackish water. Its extracellular products with antibacterial activities were shown to be stable under wide range of pH, temperature, and proteinase K. It was initially identified as Bacillus amyloliquefaciens using API identification kits and confirmed to be B. amyloliquefaciens strain (GenBank accession number DQ422953) by phylogenetic analysis. In addition, it was shown to be safe formammalians, had a wide anti-A. hydrophila spectrum, and exhibited significant effects on inhibiting the growth of the eel-pathogenic A. hydrophila both in vitro and in vivo. To the best of our knowledge, this is the first report on a promising antagonistic Bacillus amyloliquefaciens strain from brackish water sediment against eel-pathogenic A. hydrophila. [ABSTRACT FROM AUTHOR]

Background: Grass carp reovirus (GCRV) is the causative pathogen of grass carp hemorrhagic disease, one of the major diseases damaging grass carp Ctenopharyngon idellus breeding industry in China. Prevention and control of the disease is impeded largely due to the lack of research in economic subunit vaccine development. This study aimed to evaluate the potential of viral outer shell protein VP5 as subunit vaccine. Methods: The vp5 gene was isolated from the viral genome through RT-PCR and genetically engineered to express the recombinant VP5 protein in E coli. The viral origin of the recombinant protein was confirmed by Western blot analysis with a monoclonal antibody against viral VP5 protein. Polyclonal antibody against the recombinant VP5 protein was prepared from mice. A microneutralization assay was developed to test its neutralizing ability against GCRV infection in cell culture. Results: The GST-VP5 fusion protein (rVP5) was produced from E. Coli with expected molecular weight of 90 kDa. The protein was purified and employed to prepare anti-VP5 polyclonal antibody from mice. The anti-VP5 antibody was found to neutralize GCRV through in vitro microneutralization assay and viral progeny quantification analysis. Conclusions: The present study showed that the viral VP5 protein was involved in viral infection and bacterially-expressed VP5 could be suitable for developing subunit vaccine for the control of GCRV infection. [ABSTRACT FROM AUTHOR]