Thyme essential oil inhibits intracellular replication of CyHV-3 and inactivates extracellular virus. An in vitro study.

Cyprinid herpesvirus 3 (CyHV-3) can induce up to 100% mortality among carp populations. To date, there has been no safe method to prevent the consequences of the activity of CyHV-3. Thyme is widely used in cooking due to its flavour. Both thyme and thyme essential oil (TEO) are used in traditional herbal medicine, mainly to treat respiratory system disorders. In this study, TEO containing predominantly cymene and thymol was applied to explore its antiviral effect. The toxicity of TEO was examined in MTT and crystal violet assays. The anti-CyHV-3 activity of TEO in the intracellular and extracellular stages of the viral replication cycle was explored in a plaque assay and TaqMan qPCR. TEO interfered with the intracellular stages of the CyHV-3 replication cycle with selectivity indexes (SI) of around 5. It also displayed virucidal activity in a dose- and time-dependent manner. Two-hour preincubation of CyHV-3 with TEO generated SI, ranging from 13.37 to 18.47 depending on cell line and method of examination. Preincubation of cells with TEO at a safe concentration did not decrease the intracellular viral DNA copy number, which suggests that TEO does not disturb the attachment of the virus to the cells. Further research regarding the antiviral activity of compounds of TEO is required in order to indicate the most potent molecules that could be considered candidates for application in aquaculture.

Acyclovir inhibits Cyprinid herpesvirus 3 multiplication in vitro.

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is an aetiological agent of a virulent and lethal disease in common and koi carp. In this study, we examined in vitro the anti-CyHV-3 activity of acyclovir (ACV), nucleoside analogue commonly used against human herpesviruses, as well as acyclovir monophospate (ACV-MP). The cytotoxicity of the ACV and the ACV-MP for two common carp cell lines, CCB (Common carp brain) and KF1 (Koi carp fin 1), was determined by means of MTT and crystal violet assays. In subsequent studies, the concentration of 66.67 µM was applied. The ACV and the ACV-MP (66.67 µM) inhibited a cytopathic effect (CPE) induced by the CyHV-3 virus in the CCB (ACV by 66%, ACV-MP by 58%) and the KF1 (ACV by 25%, ACV-MP by 37%). The viral load measured by the means of TaqMan qPCR was reduced in a range of 67%-93% depending on the analogue, the cell line and the time of incubation. The expression of viral genes (ORF149, ORF3, ORF134 and ORF78) in CCB cells infected with the CyHV-3 was strongly downregulated within the range of 78%-91%. In summary, both the ACV and the ACV-MP can inhibit CyHV-3 replication in vitro.

The impact of manganese on biofilm development of Bacillus subtilis.

Bacterial biofilms are dynamic and structurally complex communities, involving cell-to-cell interactions. In recent years, various environmental signals that induce the complex biofilm development of the Gram-positive bacterium Bacillus subtilis have been identified. These signalling molecules are often media components or molecules produced by the cells themselves, as well as those of other interacting species. The responses can also be due to depletion of certain molecules in the vicinity of the cells. Extracellular manganese (Mn2+) is essential for proper biofilm development of B. subtilis. Mn2+ is also a component of practically all laboratory biofilm-promoting media used for B. subtilis. Comparison of complex colony biofilms in the presence or absence of supplemented Mn2+ using microarray analyses revealed that genes involved in biofilm formation are indeed downregulated in the absence of Mn2+. In addition, Mn2+ also affects the transcription of several other genes involved in distinct differentiation pathways of various cellular processes. The effects of Mn2+ on other biofilm-related traits like motility, antimicrobial production, stress and sporulation were followed using fluorescent reporter strains. The global transcriptome and morphology studies highlight the importance of Mn2+ during biofilm development and provide an overview on the expressional changes in colony biofilms in B. subtilis.

Hematological Alterations as a Response to Exposure to Selected Fungicides in Common Carp (Cyprinus carpio L.).

The aim of the present study was to assess the hematological response of common carp to fungicides and to determine recovery patterns in fungicide-free water. Fish were exposed to mancozeb, prochloraz or tebuconazole (at concentrations of 1.0, 1.0 and 2.5 mg 1⁻¹, respectively) for 14 days followed by a 30-day recovery period. The following hematological parameters were examined after 1, 3 and 14 days of exposure as well as after recovery time: red blood cells (RBC), hematocrit (Het), total hemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), total number of leukocytes (WBC) and leukograms. All analyzed parameters revealed alterations in relation to control samples. The pattern of these changes was irregular, showing either an increase or decrease at different time points of the experiment and not all observed differences were statistically significant. The most noticeable fungicide-specific changes were,observed on the 1st and 14th days of chemical exposure. The majority of the parameters under investigation returned to the control levels after a detoxication period. However, some of the exerted effects were irreversible (Hb, MCH, MCHC and WBC for fish subjected to mancozeb; Hb, MCH, MCHC and monocyte count for fish subjected to prochloraz; Hct and monocyte number for fish subjected to tebuconazole). All of the observed hematoloaical changes were not toxin-soecific.

Anti-CyHV-3 Effect of Fluorescent, Tricyclic Derivative of Acyclovir 6-(4-MeOPh)-TACV in vitro.

INTRODUCTION: Cyprinid herpesvirus 3 (CyHV-3) is a virus infecting carp with disease symptoms of gill necrosis, fish discoloration, sunken eyes, and mortality reaching 90%. Several research groups have examined how to potentially abate the consequences of viral activity. Recently we showed that acyclovir inhibits CyHV-3 replication in vitro and in the present study we examined the anti-CyHV-3 activity of the tricyclic derivative of acyclovir 6-(4-MeOPh)-TACV (T-ACV), a fluorescent molecule known for higher lipophilicity than acyclovir, and therefore potentially better candidate for application in vivo. MATERIAL AND METHODS: CCB and KF1 cell lines were incubated with T-ACV at concentrations of 0, 66.67, and 133.33 µM for three days and toxicity examined with MTT and CV assays. To investigate the antiviral activity of T-ACV, the lines were infected with CyHV-3 or mock infected and incubated for three days with the drug at concentrations of 0 or 66.67 µM. The activity of T-ACV was evaluated by plaque assay and TaqMan qPCR. RESULTS: T-ACV at a concentration of 66.67 µM displayed low toxicity and inhibited CyHV-3 activity by 13-29%, varying by cell line and method. CONCLUSION: The low anti-CyHV-3 activity of T-ACV indicates that it would be reasonable to screen several tricyclic derivatives of acyclovir for such activity.