Li-Kuang Chen

Comparison of Capture Immunoglobulin M (IgM) and IgG Enzyme-Linked Immunosorbent Assay (ELISA) and Nonstructural Protein NS1 Serotype-Specific IgG ELISA for Differentiation of Primary and Secondary Dengue Virus Infections

ABSTRACT We have found that NS1 serotype-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) can be used to differentiate primary and secondary dengue virus infections. This is due to the fact that the NS1-specific IgG antibody cannot be detected before day 9 of illness for primary infection, so the NS1-specific IgG antibodies measured in acute-phase sera must come from previous infection. Comparison of NS1 serotype-specific IgG ELISA with envelope- and membrane-specific capture IgM and IgG ELISA in the differentiation of primary and secondary dengue virus infections showed good correlation (95.90% agreement). Most important, we have found that the serotype of the dengue virus from the majority of patients with primary infection could be correctly identified when convalescent-phase or postinfection sera were analyzed by NS1 serotype-specific IgG ELISA. These findings suggested that NS1 serotype-specific IgG ELISA could be reliably applied for serodiagnosis and seroepidemiological study of dengue virus infection.

Evaluation of protective efficacy and immune mechanisms of using a non-structural protein NS1 in DNA vaccine against dengue 2 virus in mice.

To evaluate the potential of DNA vaccine against dengue (DEN) infection, we characterize the protective efficacy and immune responses of mice intramuscularly injected with plasmid encoding DEN-2 non-structural protein 1 (NS1). Intravenously challenged by lethal DEN-2, mice vaccinated with NS1-DNA exhibited a delay onset of paralysis, a marked decrease of morbidity, and a significant enhancement of survival. In addition to a moderate increase of NS1-specific antibody titer from immunized mice measured by ELISA, a strong priming effect on anti-NS1 response was also noticed in plasmid NS1-vaccinated mice by radioimmunoprecipitation (RIP) or immunoblot analysis. Interestingly, newborn mice from NS1-DNA-immunized dam showed stronger resistance to viral challenge, as compared to those from vector DNA or PBS-immunized dams, indicating the protective role of NS1-specific antibody. In contrast to humoral immune response, DNA immunization can elicit strong cellular immune responses, including NS1-specific T cell proliferation and cytolytic activity. The NS1-DNA-induced protection can be further augmented by co-injection of plasmid encoding interleukin 12 (IL-12), suggesting an effector role of Th1 immunity against DEN infection. In summary, our results suggest the potential of NS1-DNA vaccine against DEN infection, and indicate both NS1-specific humoral and cellular immune responses contribute to the protection.

Dengue NS1-specific antibody responses: isotype distribution and serotyping in patients with Dengue fever and Dengue hemorrhagic fever.

To understand the antibody responses to dengue (DEN) nonstructural 1 (NS1) glycoprotein and their roles in protective immunity or pathogenesis of dengue fever (DF) and dengue hemorrhagic fever (DHF), we have analyzed the NS1‐speccific IgM, IgA and IgG antibodies from patients with DF and DHF. An isotype‐specific, indirect enzyme‐linked immunosorbent assay (ELISA) was established by coating a NS1‐specific monoclonal antibody (MAb), D2/8‐1, to capture soluble NS1 antigens secreted in the culture supernatants of Vero cells infected with DEN virus. We observed strong anti‐NS1 antibody responses in all of the convalescent sera of patients with DF and DHF. Similar NS1‐specific isotypic and serotypic antibody responses were found in the sera from DF and DHF patients. The results showed that all DEN infections induced significant NS1‐specific IgG, whereas 75% and 60% of primary DF patients vs. 40% and 90% of secondary DF patients produced IgM and IgA antibodies, respectively. Specificity analysis showed that DEN NS1‐specific IgG and IgA antibodies cross‐react strongly to Japanese encephalitis (JE) virus NS1 glycoprotein, whereas DEN NS1‐specific IgM antibodies do not cross‐react to JE virus NS1 glycoprotein at all. The serotype specificity of NS1‐specific IgM, IgA and IgG were found to be 80%, 67% and 75% for primary infections, and 50%, 22% and 30% for secondary infections in positive samples of DF patients. Similar pattern was found in DHF patients. The results showed that all of the DF and DHF patients produced significant NS1‐specific antibodies. We did not observe direct correlation between the anti‐NS1 antibody responses and DHF because sera from patients with DF and DHF showed similar anti‐NS1 antibody responses. J. Med. Virol. 62:224–232, 2000.

Potential application of nonstructural protein NS1 serotype-specific immunoglobulin G enzyme-linked immunosorbent assay in the seroepidemiologic study of dengue virus infection: correlation of results with those of the plaque reduction neutralization test.

ABSTRACT An NS1 serotype-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to differentiate primary and secondary dengue virus infections and serotypes of primary dengue virus infection. For this report, we carried out retrospective seroepidemiologic studies on serum samples collected from residents of Liuchiu Hsiang, Pingtung County, an isolated island in southern Taiwan during 1997-1998. The results demonstrated that good correlation existed between dengue virus NS1 serotype-specific immunoglobulin G (IgG) ELISA and dengue virus plaque reduction neutralization test (PRNT). Our data suggested that NS1 serotype-specific IgG ELISA could replace PRNT for seroepidemiologic studies to differentiate Japanese encephalitis and dengue virus infections and for dengue virus serotyping.

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.

Antibody to the nonstructural protein NS1 of Japanese encephalitis virus: Potential application of MAB based indirect ELISA to differentiate infection from vaccination

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect and differentiate the antibody responses to Japanese encephalitis (JE) virus nonstructural protein NS1 between infected and vaccinated individuals. The results showed that all convalescent sera from JE patients contained NS1-specific IgG antibodies, while 65 and 40% of these sera showed detectable NS1-specific IgM and IgA antibodies, respectively. Specificity analysis showed that NS1-specific IgM and IgA antibodies from JE patients do not cross-react to dengue virus NS1 glycoprotein, while IgG antibodies from 10% of JE patients showed significant cross-reaction to dengue virus NS1 glycoprotein. To differentiate infection from vaccination, the immune sera from 24 children vaccinated with inactivated JE vaccine were analyzed. The data showed that none of these immune sera had detectable NS1-specific IgG antibodies. The results demonstrated the potential application of JE NS1-specific indirect ELISA to differentiate infection from vaccination.

Flow Cytometry Compared with Indirect Immunofluorescence for Rapid Detection of Dengue Virus Type 1 after Amplification in Tissue Culture

ABSTRACT Dengue virus (DV) was detected early in infected mosquito C6/36 cells by using indirect immunofluorescence (IF) in conjunction with flow cytometry. Three fixation-permeabilization methods and three DV serotype 1 (DEN-1)-specific monoclonal antibodies, 8-8 (anti-E), 16-4 (anti-NS1), and 15F3-1 (anti-NS1), were evaluated for the detection of DEN-1 in infected C6/36 cells. We found that these three monoclonal antibodies were capable of detecting DV in C6/36 cells as early as 24 h postinoculation by using a conventional indirect IF stain. Both 8-8 and 16-4 detected DV earlier and showed a greater number of DV-positive cells than 15F3-1. In flow cytometry, 3% paraformaldehyde plus 0.1% Triton X-100 with 16-4, the best fixation-permeabilization method for testing DV, showed higher sensitivity (up to 1 PFU) than indirect IF stain. The higher sensitivity of 16-4 in detecting DEN-1 was found with both IF and flow cytometry. Flow cytometry, which had a sensitivity similar to that of nested reverse transcription-PCR, was more sensitive in detecting DV in the infected mosquito cells 10 h earlier than the conventional IF stain. When clinical specimens were amplified in mosquito C6/36 cells and then assayed for DV using flow cytometry and conventional virus isolation at day 7 postinfection, both methods had 97.22% (35 out of 36) agreement. Moreover, among 12 positive samples which were detected by conventional culture method, the flow cytometry assay could detect DV in 58.33% (7 out of 12) of samples even at day 3 postinfection. In conclusion, both monoclonal antibodies 8-8 and 16-4 can be used for the early detection of DEN-1-infected C6/36 cells, with 16-4 (anti-NS1) being the best choice for the rapid diagnosis of DV by both the IF staining and flow cytometry methods.

Dengue Virus Serotyping Based on Envelope and Membrane and Nonstructural Protein NS1 Serotype-Specific Capture Immunoglobulin M Enzyme-Linked Immunosorbent Assays

ABSTRACT Envelope and membrane (E/M) and nonstructural protein NS1 serotype-specific capture Immunoglobulin M (IgM) enzyme-linked immunosorbent assays (ELISAs) were developed to differentiate four dengue virus serotypes. A total of 93 anti-dengue virus IgM-positive serum samples collected between days 5 and 45 of illness from 59 confirmed dengue patients were analyzed. The results showed that positive serotype specificity could be identified for 86.1 and 47.6% of serum samples tested for E/M-specific IgM antibodies versus 83.3 and 42.9% of serum samples tested for NS1-specific IgM antibodies from patients with primary and secondary dengue virus infections, respectively. Dual analyses with both E/M and NS1 serotype-specific capture IgM ELISAs showed that positive serotype specificity could be correctly identified for 98.6 and 61.9% of all of the primary and secondary serum samples tested, respectively. These findings suggested that E/M and NS1 serotype-specific capture IgM ELISAs have the potential to be of use in dengue virus serotyping.

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.

Localizations of NS3 and E proteins in mouse brain infected with mutant strain of Japanese encephalitis virus

SummaryInfection with a mutant Japanese encephalitis virus (JEV) strain RP-2ms showed reduced neurovirulence than wild type or RP-9 strains after inoculation in BALB/c mice. However, higher intracellular viral titer was detected in Rp-2ms infected cultured cells. Localizations of non-structural 3 (NS3) and envelope (E) proteins were demonstrated by immunocytochemistry. NS3 protein was primarily found in the pyramidal neurons in cerebrum, in the molecular and granular layers of cerebellum. Neither E nor NS3 protein was detected in Purkinje cells of the cerebellum. Immunoelectron microscopic observations showed that E and NS3 proteins were positive in JEV-induced membranous systems, mainly hypertrophic rough endoplasmic reticulum (rER) and membrane vesicle structure (MVS) but not smooth membrane structure. Virus particles were seen in the Golgi apparatus, rER, nuclear envelope, MVS and cytoplasmic vacuoles. Different mechanisms of intracellular trapping in vivo provide a possible basis for attenuation of RP-2ms strains of JEV.