Detection of Anti-ZIKV NS1 IgA, IgM, and Combined IgA/IgM and Identification of IL-4 and IL-10 as Potential Biomarkers for Early ZIKV and DENV Infections in Hyperendemic Regions, Thailand.

The frequency of Zika virus (ZIKV)-specific IgA and IgM and the cytokine expression profile of ZIKV-infected patients in hyperendemic areas remain unclear. This study investigated the rates of ZIKV non-structural protein 1 (NS1)-specific IgA and IgM and evaluated serum cytokine levels of ZIKV and Dengue virus (DENV) cases in Thailand to identify potential diagnostic biomarkers, elucidate the immunity against ZIKV and DENV, and investigate the association between cytokine levels and ZIKV symptoms. Low rates of positivity for ZIKV NS1-specific IgA and IgM were detected in our study. ZIKV NS1 IgA/M (11%, 11/101) in combination was more frequently detected than ZIKV NS1 IgM (2%, 2/101) or ZIKV NS1 IgA (4%, 4/96) alone, especially in acute ZIKV cases with previous DENV exposure (14%, 10/72). Cytokine analysis showed that both ZIKV and DENV infections induced polyfunctional immunity, and the latter triggered more prolonged responses. The existence of significant differences in IL-4 and IL-10 levels between acute ZIKV and acute DENV cases suggested that IL-4 (p = 0.0176) and IL-10 (p = 0.0003) may represent biomarkers for acute ZIKV and acute DENV infections, respectively. Analysis of the association between increased cytokine levels and ZIKV symptoms indicated that CXCL10 (p = 0.0029) was associated with exanthema, while IL-5 (p = 0.0496) was linked to headache. The detection of ZIKV NS1 IgA and IgM in combination may enhance the diagnosis of early ZIKV infection, particularly when levels of IgM or IgA alone are low or undetectable. IL-4 and IL-10 may serve as targets for the development of diagnostic tools to detect ZIKV and DENV infections early, respectively, in flavivirus-endemic regions.

In vitro analysis of synthetic peptides in blocking the entry of dengue virus.

Dengue fever is the most prevalent arthropod-borne viral disease, and no specific therapeutic or promising antiviral drug is available for its treatment. Peptide inhibitors are less toxic than synthetic compounds and have found proven effective against viral infections. Here, three peptides that mimic part of the E protein of the dengue virus (DENV) were synthesized and evaluated for their inhibitory activity against four serotypes of DENV in African green monkey kidney (Vero) and rhesus macaque (Macaca mulatta) monkey kidney (LLC-MK2) cell lines. The three peptides, Pep1, Pep2, and Pep3 are located in domains I, II, and III of the E protein respectively. All three peptides effectively reduced >80% of focus forming units in the virus treated mammalian cell lines than control and exhibited their IC50 in the range of 10-33 µM. Pep1 was found effective against DENV-2, DENV-3, and DENV-4 (IC80 below 50 µM). Pep2 showed the highest inhibitory activity against all four serotypes (IC50 below 20 µM). Pep3 reduced the 80% focus forming units in all serotypes at the concentration of 40 µM. Evaluation of peptides at different time points of viral infection in the mammalian cell lines revealed that the peptides inhibited viral infection by binding to the virus and not by binding to cellular receptors and blocking viral entry. The peptides assumedly exert their inhibitory effects by binding to the E protein and repressing its conformational changes; this prevents the virus from binding to cellular receptors, thereby inhibiting viral entry. Hence, these peptides might limit viral spread and reduce the virus's ability to infect.

A synthetic peptide derived from domain III envelope glycoprotein of Dengue virus induces neutralizing antibody.

Dengue virus (DENV) is an arthropod-borne human pathogen that represents a severe public health threat in both endemic and non-endemic regions. So far, there is no licensed vaccine or specific drugs available for dengue fever. A fifteen-amino-acid-long peptide that includes the NGR motif was chemically synthesized and conjugated with keyhole limpet hemocyanin. A standard immunization protocol was followed for the production of polyclonal antibodies by immunizing rabbits against the synthetic peptide. The immune response elicited high-titer polyclonal antibodies with the reactivity of the anti-peptide antibody against both synthetic peptide and four serotypes of DENV confirmed by DOT-ELISA. Neutralizing activity of anti-peptide antibody was found to be cross-reactive and effective resulting in 60% reduction of infectivity at 1:200 dilution in all four serotypes of DENV. Our findings have the potential to further improve our understanding of virus-host interactions and provide new insights into neutralizing antibodies and could also be used as a drug target.

Impairment of CD4+CD25+ regulatory T cells in C4-deficient mice.

OBJECTIVE: To investigate the association between deficiencies of early components in the classical complement pathway and the development of SLE. METHODS: Forty inbred C57BL/6J mice and 40 knockout C4 complement gene (C4KO) mice, which included 10 mice in each age group (2, 4, 6, and 8 months) were used. The enumeration of CD4+CD25+ Tregs frequencies in bone marrow, spleen and peripheral blood from both normal and C4KO groups were performed by flow cytometry. The expression levels of Foxp3 and TGF-beta in the same tested tissues were measured using real time PCR. The antinuclear antibodies (ANA) were semi-quantitatively measured using ELISA. RESULTS: We report decreased frequencies of CD4+CD25+ Tregs and reduced expression levels of Foxp3 and TGF-beta, which efficiently program the development and function of Tregs, in lymphoid tissues and peripheral blood of C4KO mice. In this study, C4KO mice have higher titers of ANA than those of normal mice. Higher frequencies of mice positive for ANA are also found in older mice. CONCLUSIONS: The deficiency of the C4 gene induces the decreased numbers of Tregs that further increase the production of ANA resulting in the development of an autoimmune disorder. The outcomes of our study help us to understand the association between the deficiency of C4 in the classical complement pathway and development of autoimmune disorder via the role of Tregs.

Leptospirosis serodiagnosis by ELISA based on recombinant outer membrane protein.

The outer membrane protein LipL21, LipL32, LipL41 and Loa22 of Leptospira interrogans serovar Copenhageni were previously revealed by immunoproteomic analysis, using sera from acute phase infection in a guinea pig. The full-length DNA of each protein was then cloned from the same serovar and expressed in pRSET vector. The obtained molecular weight (MW) of recombinant proteins rLipL21, rLipL32 and rLoa22 were slightly higher than the MW predicted from nucleotide sequences of each inserted gene, while only the N-terminal half of rLipL41 was obtained. Mice antiserum raised against each purified recombinant protein could react with the whole cell lysate of leptospiral serovars, implying that leptospiral native proteins shared a common epitope with recombinant protein. Serodiagnosis using recombinant protein antigen based on indirect ELISA procedure was developed in this study. The optimization of the ELISA components lead to determination of optical density (OD) from a single serum-dilution of 1:1000 in the leptospirosis patients group and normal healthy control group. The cut off OD values for both IgG and IgM class were investigated, and based on this fixed dilution only the IgG class could be used for differential diagnosis of patients and normal individuals. Compared with the MAT assay, ELISA assay utilizing both rLipL32 and rLoa22 as antigen, gave high accuracy and could thus be useful as a confirmative serology test.

Preliminary post-tsunami water quality survey in Phang-Nga province, southern Thailand.

This preliminary water quality survey was performed eight weeks after the tsunami hit Phang-Nga Province on 26 December 2004. Water samples collected from the affected area, 10 km parallel to the seaside, were compared with water samples from the control area approximately 4 km from the seaside, which the tsunami waves could not reach. These samples included 18 surface-water samples, 37 well-water samples, and 8 drinking-water samples, which were examined for microbiology and physical-chemical properties. The microbiological examinations focused on enteric bacteria, which were isolated by culture method, while physical-chemical properties comprised on-site testing for pH, salinity, dissolved oxygen (DO), conductivity and total dissolved solids (TDS) by portable electrochemical meter (Sens Ion 156). The results of the microbiological examinations showed that water samples in the affected areas were more contaminated with enteric bacteria than the control area: 45.4% of surface-water samples in the affected area, and 40.0% in the control; 19.0% of well-water samples in the affected area, and 7.7% in the control. All eight drinking-water samples were clear of enteric bacteria. Tests for physical-chemical properties showed that the salinity, pH, conductivity, and TDS of surface-water samples from the affected area were significantly higher than the control. The salinity, conductivity, and TDS of the well-water samples from the affected areas were also significantly greater than those from the control area. The surface and well water in the tsunami-affected area have been changed greatly and need improvement.