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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]

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]

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]

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]