Effects on viral suppression and the early-immune expression of ribavirin against spring viremia of carp virus in vitro.

Spring viremia of carp virus (SVCV) is a globally distributed virus that causes severe clinical symptoms and high mortality in fish belonging to the families Cyprinidae and Siluridae. To protect the host against viral infection, understanding the relatedness between viral susceptibility and antiviral mechanisms must be crucial. Thus, we evaluated the viral suppression efficacy of ribavirin by measuring the transcription levels of viral and immune genes in vitro. The results showed that following ribavirin treatment after SVCV infection (MOI 0.1), ribavirin inhibited SVCV replication in epithelioma papulosum cyprini (EPC) cells and completely inhibited viral gene (G and N) expression at concentrations above 10 µg/mL at 48 h post-infection. Ribavirin does not directly damage SVCV particles but inhibits early viral replication. In the absence of SVCV infection, the immunological dynamics triggered by ribavirin resulted in upregulated pattern recognition receptors and proinflammatory cytokine-related genes (i.e., PI3K, MYD88, IRAK1, RIG-І, MAVS, Mx1, TNF-α, and NF-κB). Furthermore, EPC cells treated with ribavirin following SVCV infection showed upregulation of PI3K, MYD88, IRAK1, RIG-І, TNF-α, and NF-κB genes within 24 h post-SVCV infection, suggesting that ribavirin positively inhibits the SVCV infection in vitro.

In Vitro Viral Recovery Yields under Different Re-Suspension Buffers in Iron Flocculation to Concentrate Viral Hemorrhagic Septicemia Virus Genotype IVa in Seawater.

Iron flocculation is widely used to concentrate viruses in water, followed by Fe-virus flocculate formation, collection, and elution. In the elution stage, an oxalic or ascorbic acid re-suspension buffer dissolved iron hydroxide. After the concentration of viral hemorrhagic septicemia virus (VHSV) in seawater (1 × 101 to 1 × 105 viral genome copies or plaque-forming unit (PFU)/mL), the recovery yield of the viral genome using quantitative real-time PCR (qRT-PCR) and viral infectivity using the plaque assay were investigated to evaluate the validity of the two re-suspension buffers to concentrate VHSV. The mean viral genome recovery yield with oxalic and ascorbic acid was 71.2 ± 12.3% and 81.4 ± 9.5%, respectively. The mean viral infective recovery yields based on the PFU were significantly different between the two buffers at 23.8 ± 22.7% (oxalic acid) and 4.4 ± 2.7% (ascorbic acid). Notably, although oxalic acid maintains viral infectivity over 60% at a viral concentration above 105 PFU/mL, the infective VHSVs were not sufficiently recovered at a low viral concentration (102 PFU/mL, <10%). To support this result, concentrated VHSV was inoculated in Epithelioma papulosum cyprini (EPC) cells to confirm cell viability, viral gene expression, and extracellular viral titer. All results demonstrated that oxalic acid buffer was superior to ascorbic acid buffer in preserving viral infectivity.

In vitro and in vivo evaluation of the antiviral activity of arctigenin, ribavirin, and ivermectin against viral hemorrhagic septicemia virus infection.

Viral hemorrhagic septicemia virus (VHSV) causes a severe and often lethal infection in olive flounder (Paralichthys olivaceus) in Korea, resulting in mass mortality and substantial economic loss. As a potential prevention strategy for infectious viral diseases, this study aimed to evaluate the antiviral activity of three compounds (arctigenin [ARG], ribavirin [RBV], and ivermectin [IVM]) against VHSV infection in vitro and in vivo. In epithelioma papulosum cyprini cells, the expression of both VHSV glycoprotein (G) and nucleoprotein (N) genes were significantly suppressed by the three compounds in a dose-dependent manner (P < 0.05). Also, cell morphology and viability were maintained at the following concentrations: ARG 1.5 mg/L, RBV 2.5 mg/L, and IVM 10 mg/L. The fish that were treated with RBV (8.33 mg/kg) and IVM (0.25 mg/kg) before VHSV infection and those treated with IVM (0.25 mg/kg) after VHSV infection showed significant improvements in the survival rate, a reduction in the viral shedding rate, and downregulation of viral gene expression compared to those seen in fish with naïve VHSV infections. Furthermore, among the innate immune genes studied, persistent expression of Mx and upregulation of tumor necrosis factor-α gene expression in VHSV-infected fish treated with RBV and IVM revealed that these compounds might induce an immunostimulatory effect as one of their antiviral activities. Overall, this study supports the use of RBV and IVM as antiviral agents to control VHSV infections in olive flounder.

Application of iron flocculation to concentrate white spot syndrome virus in seawater.

The iron flocculation method, which comprises the Fe-virus flocculate formation-filtration-resuspension steps, is extensively used to concentrate and precipitate viruses distributed in water. To apply this method to concentrate white spot syndrome virus (WSSV) in seawater, viral genomic and infective recovery yields were compared between polyethylene sulfone (PES) and polycarbonate (PC) membrane filters and two types of resuspension buffers (oxalate and ascorbate). Viral genome quantitation was determined above a 95 % limit of detection (11.48 viral DNA copies/µL) using quantitative real-time PCR. From WSSV-spiked seawater (100-106 viral DNA copies/mL), the viral genomic recovery yields of the PES-Oxalate, PC-Oxalate, PES-Ascorbate, and PC-Ascorbate conditions were 78.67 % ± 12.90 %, 84.53 % ± 24.30 %, 85.59 % ± 16.98 %, and 93.74 % ± 7.44 %, respectively. The detectable Fe-virus flocculates collected by the PC membrane were approximately 101 WSSV DNA copies/mL of seawater, a value more than 10-fold higher than that compared to the PES membrane filter (102 WSSV DNA copies/mL), regardless of the resuspension buffer types. WSSV resuspended with oxalate buffer caused mass mortality among whiteleg shrimp (Litopenaeus vannamei), inducing the expression of the virus envelope protein, VP28, similar to that of a native virus, suggesting stable viral activity during the resuspension process. Based on the PES-Ascorbate, WSSV particles could be successfully concentrated in seawater from shrimp farms with white spot disease outbreaks (approximately 102 WSSV DNA copies/mL). Collectively, these findings indicate that the simple and efficient method of iron flocculation is sufficient to concentrate WSSV in seawater and could be used as a non-invasive approach and one of the reasonable diagnostic processes for white spot disease surveillance.

Lipocalin 2 (Lcn2) interferes with iron uptake by Brucella abortus and dampens immunoregulation during infection of RAW 264.7 macrophages.

Lipocalin 2 (Lcn2) is an important innate immunity component against bacterial pathogens. In this study, we report that Lcn2 is induced by Brucella (B.) abortus infection and significantly contributes to the restriction of intracellular survival of Brucella in macrophages. We found that Lcn2 prevented iron uptake by B. abortus through two distinct mechanisms. First, Lcn2 is secreted to capture bacterial siderophore(s) and abrogate iron import by Brucella. Second, Lcn2 decreases the intracellular iron levels during Brucella infection, which probably deprives the invading Brucella of the iron source needed for growth. Suppression of Lcn2 signalling resulted in a marked induction of anti-inflammatory cytokine, interleukin 10, which was shown to play a major role in Lcn2-induced antibrucella immunity. Similarly, interleukin 6 was also found to be increased when Lcn2 signalling is abrogated; however, this induction was thought to be an alternative pathway that rescues the cell from infection when the effective Lnc2 pathway is repressed. Furthermore, Lcn2 deficiency also caused a marked decrease in brucellacidal effectors, such as reactive oxygen species and nitric oxide but not the phagolysosome fusion. Taken together, our results indicate that Lcn2 is required for the efficient restriction of intracellular B. abortus growth that is through limiting iron acquisition and shifting cells to pro-inflammatory brucellacidal activity in murine macrophages.

Inhibitory Effect of the Ethanol Extract of a Rice Bran Mixture Comprising Angelica gigas, Cnidium officinale, Artemisia princeps, and Camellia sinensis on Brucella abortus Uptake by Professional and Nonprofessional Phagocytes.

In this study, we evaluated the inhibitory effect of a rice bran mixture extract (RBE) on Brucella abortus pathogenesis in professional (RAW 264.7) and nonprofessional (HeLa) phagocytes. We fermented the rice bran mixture and then extracted it with 50% ethanol followed by gas chromatography-mass spectrometry to identify the components in RBE. Our results clearly showed that RBE caused a significant reduction in the adherence of B. abortus in both cell lines. Furthermore, analysis of phagocytic signaling proteins by western blot assay revealed that RBE pretreatment resulted in inhibition of phosphorylation of JNK, ERK, and p38, leading to decline of internalization compared with the controls. Additionally, the intensity of F-actin observed by fluorescence microscopy and FACS was remarkably reduced in RBE-pretreated cells compared with control cells. However, the intracellular replication of B. abortus within phagocytes was not affected by RBE. Taken together, these findings suggest that the phagocytic receptor blocking and suppressive effects of RBE on the MAPK-linked phagocytic signaling pathway could negatively affect the invasion of B. abortus into phagocytes.

The Bactericidal Effect of High Temperature Is an Essential Resistance Mechanism of Chicken Macrophage against Brucella abortus Infection.

Knowledge of avian host responses to brucellosis is critical to understanding how birds resist this infection; however, this mechanism is not well established. On the other hand, temperature has a major involvement in the physiology of living organisms, and cell death induced by heat is attributed to protein denaturation. This study demonstrates the direct bactericidal effect of a high temperature (41ºC) on Brucella abortus that resulted in the gradual reduction of intracellular bacteria and inhibited bacterial growth within avian macrophage HD11 in an increasing period of time. On the other hand, this study also revealed that high temperature does not affect the rate of bacterial uptake, as confirmed by the bacterial adherence assay. No significant difference was observed in the expression of target genes between infected and uninfected cells for both temperatures. This study suggests the susceptibility of B. abortus to bacterial death under a high temperature with an increased period of incubation, leading to suppression of bacterial growth.

Adding enzymes to improve the properties of the Korean barley Gwangmaek wort during mashing.

This study investigated the properties of the Korean barley Gwangmaek wort and compared them to those of the imported Pilsner. The reducing sugar content of Gwangmaek (52.13 mg/mL) was 13% lower than that of Pilsner. The filtration time for Gwangmaek (53 min) was 2.5 times longer than that of Pilsner. The α-amylase and amyloglucosidase treatments increased the reducing sugar content of Gwangmaek up to that of Pilsner. However, the filtration time (88min) significantly increased after the enzyme treatment. In terms of ß-glucan contents measured during the mashing process of Pilsner, Gwangmaek, and α-amylase and amyloglucosidase-treated Gwangmaek, the enzyme-treated Gwangmaek had the highest at 232.23mg/L, followed by Gwangmaek (190.31 mg/L) and Pilsner (82.82mg/L). When ß-glucanase was added during mashing, there was no change in reducing sugar content. However, the filtration time significantly decreased from 88 to 29 min, and viscosity also declined from 1.78 to 1.42 cp.

Is confrontation naming performance in Alzheimer's disease the nominal linguistic retrogenesis of normal development?

We investigated confrontation naming performance of patients with Alzheimer's disease (AD) and normal children (NC) to see if the nature of naming performance of AD patients is the reversal of that in normal development. Sixty items of the Boston Naming Test were given to 78 AD patients (and 40 age- and education-matched normal elderly) and 1,080 NC (3- to 14-year-olds). The analyses revealed that, firstly, the naming abilities of the AD patients demonstrated an inverse relationship with those of the NC. Secondly, from the clinical point of view, AD patients tended to lose vocabulary acquired later first while maintaining those acquired in earlier stages of development. Based on the findings, we claimed that this phenomenon was 'a nominal retrogenesis' in which 'retrogenesis' is 'the process by which degenerative mechanisms reverse the order of acquisition in normal development' as defined by Reisberg and colleagues.