Yue-Ling Huang

Identification of B-Cell Epitope of Dengue Virus Type 1 and Its Application in Diagnosis of Patients

ABSTRACT Using a serotype-specific monoclonal antibody (MAb) of dengue virus type 1 (DEN-1), 15F3-1, we identified the B-cell epitope of DEN-1 from a random peptide library displayed on phage. Fourteen immunopositive phage clones that bound specifically to MAb 15F3-1 were selected. These phage-borne peptides had a consensus motif of HxYaWb (a = S/T, b = K/H/R) that mimicked the sequence HKYSWK, which corresponded to amino acid residues 111 to 116 of the nonstructural protein 1 (NS1) of DEN-1. Among the four synthetic peptides corresponding to amino acid residues 110 to 117 of the NS1 of DEN-1, -2, -3, and -4, only one peptide, EHKYSWKS (P14M) of DEN-1, was found to bind to 15F3-1 specifically. Furthermore, P14M was shown to inhibit the binding of phage particles to 15F3-1 in a competitive inhibition assay. Histidine111 (His111) was crucial to the binding of P14M to 15F3-1, since its binding activity dramatically reduced when it changed to leucine111 (Leu111). This epitope-based peptide demonstrated its clinical diagnostic potential when it reacted with a high degree of specificity with serum samples obtained from both DEN-1-infected rabbits and patients. Based on these observations, our DEN-1 epitope-based serologic test could be useful in laboratory viral diagnosis and in understanding the pathogenesis of DEN-1.

Salicylates Inhibit Flavivirus Replication Independently of Blocking Nuclear Factor Kappa B Activation

ABSTRACT Flaviviruses comprise a positive-sense RNA genome that replicates exclusively in the cytoplasm of infected cells. Whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. Flavivirus infections quickly activate nuclear factor kappa B (NF-κB), and salicylates have been shown to inhibit NF-κB activation. In this study, we investigated whether salicylates suppress flavivirus replication and virus-induced apoptosis in cultured cells. In a dose-dependent inhibition, we found salicylates within a range of 1 to 5 mM not only restricted flavivirus replication but also abrogated flavivirus-triggered apoptosis. However, flavivirus replication was not affected by a specific NF-κB peptide inhibitor, SN50, and a proteosome inhibitor, lactacystin. Flaviviruses also replicated and triggered apoptosis in cells stably expressing IκBα-ΔN, a dominant-negative mutant that antagonizes NF-κB activation, as readily as in wild-type BHK-21 cells, suggesting that NF-κB activation is not essential for either flavivirus replication or flavivirus-induced apoptosis. Salicylates still diminished flavivirus replication and blocked apoptosis in the same IκBα-ΔN cells. This inhibition of flaviviruses by salicylates could be partially reversed by a specific p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Together, these results show that the mechanism by which salicylates suppress flavivirus infection may involve p38 MAP kinase activity but is independent of blocking the NF-κB pathway.

A highly attenuated strain of Japanese encephalitis virus induces a protective immune response in mice.

A pair of virulent (RP-9) and attenuated (RP-2ms) mutants of Japanese encephalitis virus (JEV) were generated from a Taiwanese isolate NT109. The mutants differed in several aspects in vitro and in vivo. RP-2ms exhibited smaller plaque than RP-9 on BHK-21 cells, and when intracerebrally injected, RP-2ms was much less neurovirulent than RP-9. As peripherally inoculated, RP-2ms lost neuroinvasiveness while RP-9 penetrated blood-brain barrier, replicated in mouse brain, and killed all the mice. Single RP-2ms immunization completely protected C3H and ICR mice from a lethal challenge with RP-9; the sera from such mice contained antibodies against JEV envelope and nonstructural 1 proteins, indicating RP-2ms had replicated in the mice Neutralizing activity against NT109 in such sera was further demonstrated by plaque reduction neutralization test. In addition, significant lymphoproliferation was detected in spleen cells from the RP-2ms-immunized mice, and cytotoxic activity in these cells specific for the MHC-matched, JEV-infected cells, but not mock cells, was also observed. Altogether, these results demonstrate that RP-2ms, a highly attenuated JEV strain, can induce a protective immunity in mice.

Response of amoeboid and differentiating ramified microglia to glucocorticoids in postnatal rats: a lectin histochemical and ultrastructural study.

After glucocorticoid injection(s), the number of amoeboid microglial cells (AMC) in the corpus callosum labelled by lectin was markedly reduced when compared with the corresponding control rats. In rats killed at the age of 7 days, all the labeled cells differentiated to become ramified microglia. Ultrastructurally, the AMC in glucocorticoid-injected rats were extremely vacuolated and showed increased lipid droplets. Furthermore, the cells displayed varied lectin labelling patterns especially at both the trans saccules of the Golgi apparatus and lysosomes. In differentiating ramified microglia, massive cellular debris and lectin-stained vesicles or vacuoles were observed; some of the latter appeared to fuse with the plasma membrane. The most striking feature after glucocorticoid (GCC) treatment was the complete diminution of lectin labelling at the Golgi saccules in some differentiating ramified microglia. The present results have demonstrated different effects of glucocorticoids on AMC and differentiating ramified microglia. The differential response of AMC and differentiating ramified microglia to the immunosuppressive drugs may be attributed to the fact that these cells in the postnatal brains subserve different functions or that they are at different differentiation stages. In other words, the sensitivity of microglial cells to the immunosuppressive drugs is dependent upon the stage of cell maturation/differentiation.