Neutralizing Antibody Induction and Cytotoxic T Lymphocyte Response to Nakayama-NIH and Beijing-1 as Japanese Encephalitis Virus Vaccine Strains

The Japanese encephalitis virus (JEV), a member of the Flaviviridae family and Flavivirus genus, is transmitted by mosquitoes. JEV, of which some 35,000 cases are recorded every year, is a positive RNA virus. Two types of JEV vaccines have been developed to prevent the onset of encephalitis in humans, namely formalin-inactivated and liveattenuated vaccines. JEV inactivated vaccines are usually made using the Nakayama-NIH or Beijing-1 strains of the JEV virus. In this study, the immunological response to the Nakayama-NIH and Beijing-1 strains was analyzed as part of the effort to compile basic data which could lead to the selection of a suitable vaccine strain. To this end, the virus titer of Beijing-1 was found to be two-fold higher than that of Nakayama-NIH by plaque assay. Moreover, Beijing-1-induced neutralizing antibodies showed a higher level of titers when confronted by Korean JEV isolates than Nakayama-NIH-induced neutralizing antibodies (1:320 vs. 1:160, respectively). However, as a minimum ratio of 1:10 neutralizing antibody titers are required to protect against JEV infection, both strains in effect exhibited a sufficient level of neutralizing antibody titers. What's more, Beijing-1 was found to induce a somewhat higher cytotoxic T lymphocyte (CTL) response than Nakayama-NIH. Taken together, this can be taken to mean that Beijing-1 may in fact be a more effective vaccine candidate strain when it comes to inducing a high level of protective immunity against JEV infection.

Host Gene Profiling of Coxsackievirus B3 H3- and 10A1-infected Mouse Heart

Coxsackievirus B3 (CVB3) is a non-enveloped virus that has a single-stranded RNA genome. CVB3 induces myocarditis, and ultimately, dilated cardiomyopathy. A myocarditis variant of CVB3 (CVB3 H3) and its antibody-escape mutant (CVB3 10A1) were studied previously; H3 was found to induce myocarditis and 10A1 was found to be attenuated in infected mice. Although amino acid residue 165, located in a puff region of VP2, was found to be altered (i.e., the H3 asparagine was altered to aspartate in 10A1), the detailed mechanism of attenuation was not clearly elucidated. Here, DNA microarray technology was used to monitor changes in mRNA levels of infected mouse hearts after CVB3 H3 and 10A1 infection. This tool was used to elucidate the pathogenic mechanisms of viral infection by understanding virus-host interactions. We identified several genes, including protein tyrosine kinases, Ddr2 and Ptk2, as well as Clqb and Crry, involved in complement reactions, which may be involved in these viral processes. Thus, gene profiling can provide an opportunity to understand host immune responses to viral infection for gene therapy and may contribute to the identification of the target gene that is modified during treatment of viral myocarditis.

Development of Peptide Antibody against Coxsackievirus B3 VP2

Coxsackievirus B3 (CVB3) is the nonenveloped virus containing a single-stranded positive-sense RNA as a genome. CVB3 infection can induce acute myocarditis and dilated cardiomypathy. CVB3 of icosahedral symmetry has four capsid proteins called VP1, VP2, VP3, and VP4. Although VP1 is a major antigenic determinant, VP2 is also an important protein for viral physiology, such as maturation cleavage and attenuation. However, VP2 study has been hampered, partly because VP2 antibody is not available. In this study, we developed peptide-based polyclonal VP2 antibody and analyzed its potency by Western blotting analysis and immunofluorescent assay. Purified B3-1 antibody (VP2 peptide antibody developed in here) showed the sensitivity and specificity, similar to VP1 monoclonal antibody which is commercially available. Moreover, this peptide antibody may be useful for double-staining with other antibodies derived from mouse. Therefore, the VP2 antibody may allow us to study CVB assembly and understand VP2 function in depth.

Heat Treatment Suppressed the Expression and Activity of Matrix Metalloproteinases in the Primary Cultured Astrocytes / 대한해부학회지

Matrix metalloproteinases (MMPs), which is a kind of Zn-dependent enzyme, are a family of proteolytic enzymes that can degrade the extracellular matrix and then play important roles in the pathophysiology of ischemia/reperfusion, especially MMP-2 and MMP-9. This study was aimed to demonstrate how heat treatment and Hsp70 regulate expression of MMP-2 and MMP-9. The expression and regulation of MMPs after ischemic-like injury with and without heat treatment were investigated in the astrocytes expressing Hsp70 or LacZ and mock infected cells. Primary astrocyte cultures were prepared from ICR mice and infected with retroviral vectors overexpressed Hsp70 or LacZ. After heat treatment, expression of MMP-2 mRNAs was not remarkably changed in heat moc cells. Otherwise, expression of MMP-9 mRNAs was significantly reduced by heat treatment. Expression of MMP-2 and MMP-9 in astrocytes, which were treated with ischemic injury after heat treatment, was obviously decreased than in untreated Moc cells. Moreover, Hsp70s were significantly synthesized in response to heat treatment. Compared to amount of protein expression of MMP-2 and MMP-9, the expression of MMP-2 in astrocytes expressing Hsp70, LacZ and mock infected were decreased after heat treatment, especially pro-form of protein expression of MMP-2. However, the expression of MMP-9 were decreased only in the astrocytes expressing Hsp70 by heat treatment and OGD injury after heat treatment, not shown a big change in the LacZ cells and mock infected cells. The results of activity study with MMPs protein were that MMP-2 protein activity was reduced but not in condition of ischemic injury after heat treatment. On the other hand, the activity of MMP-9 after heat treatment was similar to the results of activity of MMP-9 adding ischemic injury. In this study, we shown that Hsp70 overexpression following heat treatment reduced the expression of proMMP-2, proMMP-9 and processed MMP-2, especially. This findings can suggest a possible role of Hsp70 induced by heat treatment for regulation of MMPs and neuroprotection in ischemic injury.

Role of crosslinked protein in lung injury following total body irradiation and bone marrow transplantation

The aberrant protein crosslinks formation during lung injury as results total body irradiation (TBI) and bone marrow transplantation (BMT) therapy has been examined as apossible contributory factor in organ or tissue pathogenesis. Female C3HeB/ FeJ mice were used for an experimental animal. Carbon monoxide uptake (V(CO)) was measured at 1, 2, 3, 4 and 5 months after TBI at respective doses of 12, 14, 16 and 18 Gy 16 h prior to syngeneic BMT. Also as a measure of aberrant protein crosslinking in the inured tissues, transglutaminase (TGase)-activities and crosslinked protein were examined along with thrombin, a protease known to activate TGases. Reductions of VCO were detected following TBI and BMT. Activities of thrombin and TGase 1, and crosslinked protein in bronchoalveolar lavage (BAL) fluid of the mice 1 wk after TBI at 12 Gy and BMT were identified and found to be elevated in the treated animals. These findings suggest that elevated levels of crosslinked proteins and TGase I in the bronchoalveolar larvage during the lung injury could have enhanced the organ pathogenesis following TBI and BMT.