Identification of a Common Epitope between Enterovirus 71 and Human MED25 Proteins Which May Explain Virus-Associated Neurological Disease

Enterovirus 71 (EV71) is a major causative pathogen of hand, foot and mouth disease with especially severe neurologic complications, which mainly account for fatalities from this disease. To date, the pathogenesis of EV71 in the central neurons system has remained unclear. Cytokine-mediated immunopathogenesis and nervous tissue damage by virus proliferation are two widely speculated causes of the neurological disease. To further study the pathogenesis, we identified a common epitope (co-epitope) between EV71 VP1 and human mediator complex subunit 25 (MED25) highly expressed in brain stem. A monoclonal antibody (2H2) against the co-epitope was prepared, and its interaction with MED25 was examined by ELISA, immunofluorescence assay and Western blot in vitro and by live small animal imaging in vivo. Additionally, 2H2 could bind to both VP1 and MED25 with the affinity constant (Kd) of 10−7 M as determined by the ForteBio Octet System. Intravenously injected 2H2 was distributed in brain stem of mice after seven days of EV71 infection. Interestingly, 2H2-like antibodies were detected in the serum of EV71-infected patients. These findings suggest that EV71 infection induces the production of antibodies that can bind to autoantigens expressed in nervous tissue and maybe further trigger autoimmune reactions resulting in neurological disease

Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay.

The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens

Optimization of the components in NLFOA.

<p>The NLFOA conditions were optimized for (A) nanoparticle-streptavidin, (B) bio-TurboNuclease, (C) FLOS and (D) diameters of the nanoparticles. Both nanoparticle-streptavidin and bio-TurboNuclease were prepared at 1:10 serial dilutions between 10^–8 -10^-13 M. The FLOS oligos were prepared at 1:2 serial dilutions between 400–3.1 ×10^–8 M. Four different diameter sizes (13, 30, 40 and 50 nm) were analyzed for nanoparticles. Negative control (NC) contained all components except what was to be optimized. Each of the component was analyzed in five repeats and were performed in two independent experiments. Means ± SD are shown.</p

Comparison of the detection limit between NLFOA and the conventional ELISA.

<p>The detection limit for HIV-1 p24 antigen diluted in PBS by NLFOA (A) and conventional ELISA (B). The detection limit of HIV-1 p24 antigen spiked in sera from a healthy human by NLFOA (C) and conventional ELISA (D). The dotted line indicates the cutoff value that is 2.1-fold of that of the negative control (NC). Experiments with p24 in PBS (A and B) were done in five repeats and experiments with p24 in sera (C and D) were done in eight repeats. Each assay was performed in three independent experiments. Means ± SD are shown.</p

Identification of Luteolin as Enterovirus 71 and Coxsackievirus A16 Inhibitors through Reporter Viruses and Cell Viability-Based Screening

Hand, foot and mouth disease (HFMD) is a common pediatric illness mainly caused by infection with enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The frequent HFMD outbreaks have become a serious public health problem. Currently, no vaccine or antiviral drug for EV71/CA16 infections has been approved. In this study, a two-step screening platform consisting of reporter virus-based assays and cell viability‑based assays was developed to identify potential inhibitors of EV71/CA16 infection. Two types of reporter viruses, a pseudovirus containing luciferase-encoding RNA replicons encapsidated by viral capsid proteins and a full-length reporter virus containing enhanced green fluorescent protein, were used for primary screening of 400 highly purified natural compounds. Thereafter, a cell viability-based secondary screen was performed for the identified hits to confirm their antiviral activities. Three compounds (luteolin, galangin, and quercetin) were identified, among which luteolin exhibited the most potent inhibition of viral infection. In the cell viability assay and plaque reduction assay, luteolin showed similar 50% effective concentration (EC50) values of about 10 μM. Luteolin targeted the post-attachment stage of EV71 and CA16 infection by inhibiting viral RNA replication. This study suggests that luteolin may serve as a lead compound to develop potent anti-EV71 and CA16 drugs

Increased sensitivity with nanoparticle-streptavidin and FLOS.

<p>The signals generated by the nanoparticle-streptavidin and streptavidin systems were compared among four different enzyme-substrate systems: (A) TurboNuclease and FLOS; (B) alkaline phosphatase (ALP) and p-nitrophenylphosphate (pNPP); (C) horseradish peroxidase (HRP) and o-Phenylenediamine (OPD); and (D) alkaline phosphatase (ALP) and 4-methylumbelliferyl phosphate (4-MUP). Each enzyme was prepared at 1:10 serial dilutions (10^–4–10^–9 M). Negative control (NC) contained all components except the enzyme. The cutoff (2.1 fold higher than the NC signal) is indicated by the dotted line. Means ± SD are shown.</p

Determination of relative activity of different nucleases.

<p>(A) The nuclease activity was determined by mixing FLOS with serially diluted enzymes (1:10). The mean fluorescence intensity (MFI) was measured every 5 minutes. (B) Analysis of the enzyme contents in the commercial enzyme stocks by SDS-PAGE. Equal volume (10 μl) of each commercial nuclease was loaded onto a SDS-PAGE gel for electrophoresis. Exonuclease III and TurboNuclese were diluted at 1:100 and 1:10, respectively, due to the high protein concentration in the samples. (C) Determination of enzyme relative activity. The relative activity of each enzyme was defined by the positive MFI (2.1 folds above the background) at the lowest concentration of the enzyme (MFI/ng). All experiments were independently performed three times and results from one representative experiment are shown. Negative control (NC) contained all components except the enzyme. Means ± SD are shown.</p

A porcine circovirus type 2 (PCV2) mutant with 234 amino acids in capsid protein showed more virulence in vivo, compared with classical PCV2a/b strain.

BACKGROUND: Porcine circovirus type 2 (PCV2) is considered to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which has become a serious economic problem for the swine industry worldwide. The major genotypes, PCV2a and PCV2b, are highly prevalent in the pig population and are present worldwide. However, another newly emerging PCV2b genotype mutant, which has a mutation in its ORF2-encoded capsid protein, has been sporadically present in China, as well as in other countries. It is therefore important to determine the relative virulence of the newly emerging PCV2b genotype mutant, compared with the existing PCV2a and PCV2b genotypes, and to investigate whether the newly emerging mutant virus induces more severe illness. METHODOLOGY/PRINCIPAL FINDINGS: Twenty healthy, 30-day-old, commercial piglets served as controls or were challenged with PCV2a, PCV2b and the newly emerging mutant virus. A series of indexes representing different parameters were adopted to evaluate virulence, including clinical signs, serological detection, viral load and distribution, changes in immune cell subsets in the peripheral blood, and evaluation of pathological lesions. The newly emerging PCV2 mutant demonstrated more severe signs compatible with PMWS, characterized by wasting, coughing, dyspnea, diarrhea, rough hair-coat and depression. Moreover, the pathological lesions and viremia, as well as the viral loads in lymph nodes, tonsils and spleen, were significantly more severe (P<0.05) for piglets challenged with the newly emerging mutant compared with those in the groups challenged with PCV2a and PCV2b. In addition, a significantly lower average daily weight gain (P<0.05) was recorded in the group challenged with the newly emerging PCV2 mutant than in the groups challenged with the prevailing PCV2a and PCV2b. CONCLUSIONS: This is believed to be the first report to confirm the enhanced virulence of the newly emerging PCV2 mutant in vivo

Enhanced precision with NLFOA.

<p>The signal/noise (S/N) ratios were determined for both NLFOA and ELISA assays at different p24 concentrations.</p