Novel Naturally Occurring Mutations of Enterovirus 71 Associated With Disease Severity.

Infection with the re-emerging enterovirus 71 (EV-A71) is associated with a wide range of disease severity, including herpangina, encephalitis, and cardiopulmonary failure. At present, there is no FDA-approved therapeutics for EV-A71. Early diagnosis for the high-risk children is the key to successful patient care. We examined viral genome sequences at the 5' untranslated region (UTR) and the capsid protein VP1 from 36 mild and 27 severe cases. We identified five EV-A71 mutations associated with severe diseases, including (1) the 5' UTR mutations C580U, A707G, C709U; (2) a VP1 alanine-to-threonine mutation at position 280 (280T), and (3) a VP1 glutamic acid-to-(non-glutamic acid) at position 145 [145(non-E)]. SCARB2 is a known entry receptor for EV-A71. Based on a recent cryoEM structure of the EV-A71-SCARB2 binding complex, VP1-280T is near the binding interface between the VP1-VP2 complex and its entry receptor SCARB2. A de novo created hydrogen bonding between the mutant VP1-280T and the VP2-139T, could help strengthen a web-like interaction structure of the VP1-VP2 complex. A stabilized loop turn of VP2, once in contact with SCARB2, can enhance interaction with the host SCARB2 receptor for viral entry. Our findings here could facilitate early detection of severe cases infected with EV-A71 in clinical medicine. In addition, it opens up the opportunity of functional studies via infectious cDNA cloning, site-directed mutagenesis, and animal models in the future.

Immunodeficient mouse models with different disease profiles by in vivo infection with the same clinical isolate of enterovirus 71.

UNLABELLED: Like poliovirus infection, severe infection with enterovirus 71 (EV71) can cause neuropathology. Unlike poliovirus, EV71 is often associated with hand-foot-and-mouth disease (HFMD). Here we established three mouse models for experimental infection with the same clinical isolate of EV71. The NOD/SCID mouse model is unique for the development of skin rash, an HFMD-like symptom. While the NOD/SCID mice developed limb paralysis and death at near-100% efficiency, the gamma interferon receptor knockout (ifngr KO) and stat-1 knockout mice exhibited paralysis and death rates near 78% and 30%, respectively. Productive infection with EV71 depends on the viral dose, host age, and inoculation route. Levels of infectious EV71, and levels of VP1-specific RNA and protein in muscle, brain, and spinal cord, were compared side by side between the NOD/SCID and stat-1 knockout models before, during, and after disease onset. Spleen fibrosis and muscle degeneration are common in the NOD/SCID and stat-1 knockout models. The main differences between these two models include their disease manifestations and cytokine/chemokine profiles. The pathology of the NOD/SCID model includes (i) inflammation and expression of viral VP1 antigen in muscle, (ii) increased neutrophil levels and decreased eosinophil and lymphocyte levels, and (iii) hair loss and skin rash. The characteristic pathology of the stat-1 knockout model includes (i) a strong tropism of EV71 for the central nervous system, (ii) detection of VP1 protein in the Purkinje layer of cerebellar cortex, pons, brain stem, and spinal cord, (iii) amplification of microglial cells, and (iv) dystrophy of intestinal villi. Our comparative studies on these new models with oral or intraperitoneal (i.p.) infection underscored the contribution of host immunity, including the gamma interferon receptor, to EV71 pathogenesis. IMPORTANCE: In the past decade, enterovirus 71 (EV71) has emerged as a major threat to public health in the Asia-Pacific region. Disease manifestations include subclinical infection, common-cold-like syndromes, hand-foot-and-mouth disease (HFMD), uncomplicated brain stem encephalitis, severe dysregulation of the autonomic nerve system, fatal pulmonary edema, and cardiopulmonary collapse. To date, no effective vaccine or treatment is available. A user-friendly and widely accessible animal model for researching EV71 infection and pathogenesis is urgently needed by the global community, both in academia and in industry.

A new selective colorimetric and fluorescent chemodosimeter for HSO4(-) based on hydrolysis of Schiff base.

Two new receptors 1 and 2 were prepared, and their chromogenic and fluorogenic behaviors toward various anions were investigated. Receptors 1 and 2 show exclusive response toward HSO4(-) ion and also distinguish HSO4(-) from other anions by different color changes in aqueous solution (CH3CN/H2O=4/1, v/v). Between them receptor 1 selectively exhibits a pronounced HSO4(-)-induced fluorescence enhancement. The detection limit for the HSO4(-) ion was determined as (0.24±0.03µM). Thus, the receptor 1 can be used as a colorimetric and fluorescent sensor for the determination of HSO4(-) ion. The sensing mechanism has been suggested to proceed via a hydrolysis process. The hydrolysis product has been isolated and further identified by (1)H NMR spectroscopy, ESI-MS analysis and X-ray diffraction.

Human infection with avian influenza A H6N1 virus: an epidemiological analysis.

BACKGROUND: Avian influenza A H6N1 virus is one of the most common viruses isolated from wild and domestic avian species, but human infection with this virus has not been previously reported. We report the clinical presentation, contact, and environmental investigations of a patient infected with this virus, and assess the origin and genetic characteristics of the isolated virus. METHODS: A 20-year-old woman with an influenza-like illness presented to a hospital with shortness of breath in May, 2013. An unsubtyped influenza A virus was isolated from her throat-swab specimen and was transferred to the Taiwan Centres for Disease Control (CDC) for identification. The medical records were reviewed to assess the clinical presentation. We did a contact and environmental investigation and collected clinical specimens from the case and symptomatic contacts to test for influenza virus. The genomic sequences of the isolated virus were determined and characterised. FINDINGS: The unsubtyped influenza A virus was identified as the H6N1 subtype, based on sequences of the genes encoding haemagglutinin and neuraminidase. The source of infection was not established. Sequence analyses showed that this human isolate was highly homologous to chicken H6N1 viruses in Taiwan and had been generated through interclade reassortment. Notably, the virus had a G228S substitution in the haemagglutinin protein that might increase its affinity for the human α2-6 linked sialic acid receptor. INTERPRETATION: This is the first report of human infection with a wild avian influenza A H6N1 virus. A unique clade of H6N1 viruses with a G228S substitution of haemagglutinin have circulated persistently in poultry in Taiwan. These viruses continue to evolve and accumulate changes, increasing the potential risk of human-to-human transmission. Our report highlights the continuous need for preparedness for a pandemic of unpredictable and complex avian influenza. FUNDING: Taiwan Centres for Disease Control.

Synthesis of alanine-based colorimetric sensors and enantioselective recognition of aspartate and malate anions.

Two chiral colorimetric sensors (1,2) were synthesized and characterized by spectroscopic techniques and their enantioselective recognition of chiral dicarboxylic anions (D/L-aspartate and D/L-malate) was examined by UV-vis and (1)H NMR spectroscopy. Interaction of the receptors 1 and 2 with the enantiomers of aspartate or malate caused different color changes, and they act as optical chemosensors for the recognition of D-aspartate vs. L-aspartate and d-malate vs.l-malate. Receptor 1 exhibits high enantioselective binding for aspartate anions [K(A(D))/K(A(L)) = 12.15].

A new selective colorimetric and fluorescent sensor for Hg(2+) and Cu(2+) based on a thiourea featuring a pyrene unit.

A new artificial receptor 1 was developed, and its chromogenic and fluorogenic behaviors toward various metal ions were investigated. Receptor 1 shows exclusively responses toward Hg(2+) and Cu(2+) ions. It selectively senses Hg(2+) and Cu(2+) ions through two different color changes in aqueous solution (DMSO/H(2)O=4/1) and also distinguishes them from other metal ions. In addition, receptor 1 also exhibits pronounced enhancements of the fluorescence, which can visually be discernible by an orchid fluorescence in the presence of Hg(2+) ions and a strong blue fluorescence in the presence of Cu(2+) ions.

Synthesis of colorimetric sensors for isomeric dicarboxylate anions: selective discrimination between maleate and fumarate.

Four new colorimetric receptors (1-4) were synthesized and characterized. Upon addition of maleate to receptor in DMSO, the appearance of the solution of receptor 1 showed a color change from dark-blue to dark-red, which can be detected by the naked eye at parts per million. Similar experiments were repeated using receptors 2-4; the solution showed a distinct color change from blue to violet for receptor 2 and from blue-green to purple for both receptors 3 and 4, when they are formed as complexes with maleate. The striking color changes are thought to be due to the deprotonation of the thiourea moiety of the 4-nitronaphthyl chromophore. Whereas, in the addition of fumarate to receptors 1-4, the color of the solution changed from dark-blue to bright yellow for receptor and did not induce any color change for receptors 2-4. Thus, for a distinct color change, receptors 1-4 can act as optical chemosensors for recognition of maleate versus fumarate. Especially, only receptor 1 has a unique color change for the recognition of fumarate, accordingly it can be used for detection of the fumarate anion. In this research it was also found that the performance of the receptor is highly dependent on the substituent group on the phenyl ring; a stronger electron-withdrawing group resulted in a receptor with a higher binding constant with the maleate anion.

Massive expansion of EBV+ monoclonal T cells with CD5 down regulation in EBV-associated haemophagocytic lymphohistiocytosis.

Haemophagocytic lymphohistiocytosis (HLH) comprises primary and secondary forms; the secondary form is most commonly triggered by the Epstein-Barr virus (EBV; EBV-HLH). Patients with EBV-HLH usually exhibit oligoclonal or monoclonal T cell proliferation, which may mimic T cell lymphoproliferative disorder (T-LPD). This article reports on EBV-HLH in a 17-month-old girl with an extreme surge of reactive T lymphocytosis (absolute count 167x10(9)/l) with CD5 down regulation. Bone marrow aspirate and trephine contained florid haemophagocytosis and massive infiltration of CD3+ Epstein-Barr virus-encoded RNA+ lymphocytes, as seen by double labelling. These lymphocytes were monoclonal for EBV and T cell receptor gamma chain gene rearrangement. The patient responded dramatically to intravenous immunoglobulin, interferon alpha2b, ganciclovir and prednisolone, suggesting restoration of her immune system and eradication of the clonal T cells through these immunoregulatory agents. Thus, careful clinicopathological correlation is warranted in the interpretation of immunophenotyping and clonality data in T cell proliferation in association with EBV-HLH to avoid erroneous diagnosis of T-LPD.

Microchip, reverse transcription-polymerase chain reaction and culture methods to detect enterovirus infection in pediatric patients.

BACKGROUND: Enterovirus infection usually presents with mild and self-limited illness in children. However, Enterovirus type 71 can be characterized by neurotropism and may cause severe illness or even sudden death. Early detection of the virus will allow a physician to provide intensive or aggressive intervention. The purpose of the present study was to compare sensitivity of two innovative laboratory methods, that is, the DR.EV microchip method (DR. Chip Biotechnology, Shin-Tsu, Taiwan) and the reverse transcription-polymerase chain reaction (RT-PCR) method following conventional virus culture in detecting enterovirus infection in pediatric patients with herpangina or hand-foot-mouth disease. METHODS: A total of 87 children (age range: 1-8 years) were enrolled because of typical clinical findings of herpangina and hand-foot-mouth disease. Two hundred children selected after a careful clinical history review and physical examinations, were included as controls. All of these children had at least throat swab and rectal swab specimens taken and tested for evidence of enterovirus infection by microchip, RT-PCR and virus culture methods. In addition, 21 patients also had cerebrospinal fluid (CSF) specimens taken to test for possible central nervous system involvement. RESULT: The test results obtained from the 200 healthy kindergarten children were all negative for enteroviral infection by these three methods. Among the 87 test patients, the positive rates for throat swab, rectal swab and CSF by DR.EV chip, RT-PCR and virus culture were 71%, 68%, and 45% (throat swab); 66%, 61%, and 33% (rectal swab); and 52%, 29%, and 5% (CSF), respectively. There was no significant difference in the positive rates between the DR.EV chip and the RT-PCR methods (P > 0.1) on all types of specimens. However, statistically significant differences in positive rates were noted between the DR.EV chip and the conventional virus culture methods on all types of specimens (P < 0.001). Sensitivity of the microchip, RT-PCR and virus culture methods, was 82%, 72%, and 53%, respectively. CONCLUSION: The DR.EV chip method yielded a statistically higher positive rate and faster test results than the conventional viral culture method.