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Find video protocols related to scientific articles indexed in Pubmed.
Ambient Method for the Production of an Ionically Gated Carbon Nanotube Common Cathode in Tandem Organic Solar Cells.
J Vis Exp
PUBLISHED: 11-20-2014
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A method of fabricating organic photovoltaic (OPV) tandems that requires no vacuum processing is presented. These devices are comprised of two solution-processed polymeric cells connected in parallel by a transparent carbon nanotubes (CNT) interlayer. This structure includes improvements in fabrication techniques for tandem OPV devices. First the need for ambient-processed cathodes is considered. The CNT anode in the tandem device is tuned via ionic gating to become a common cathode. Ionic gating employs electric double layer charging to lower the work function of the CNT electrode. Secondly, the difficulty of sequentially stacking tandem layers by solution-processing is addressed. The devices are fabricated via solution and dry-lamination in ambient conditions with parallel processing steps. The method of fabricating the individual polymeric cells, the steps needed to laminate them together with a common CNT cathode, and then provide some representative results are described. These results demonstrate ionic gating of the CNT electrode to create a common cathode and addition of current and efficiency as a result of the lamination procedure.
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Recombinant ESAT-6-like proteins provoke protective immune responses against invasive Staphylococcus aureus disease in a murine model.
Infect. Immun.
PUBLISHED: 11-05-2014
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Staphylococcus aureus (S. aureus) is a common pathogen found in the community and in hospitals. Most notably, Methicillin-resistant S. aureus is resistant to many antibiotics, which is a growing public health concern. The emergence of drug-resistant strains has prompted the search for alternative treatments such as immunotherapeutic approaches. To date, most clinical trials of vaccines or passive immunization against S. aureus have ended in failure. In this study, we investigated two ESAT-6-like proteins secreted by S. aureus, SaEsxA and SaEsxB, as possible targets for a vaccine. Mice vaccinated with these purified proteins elicited high titers of anti-SaEsxA and anti-SaEsxB antibodies, but these antibodies could not prevent S. aureus infection. On the other hand, rSaEsxA and rSaEsxB could induce Th1- and Th17-biased immune responses in mice. Mice immunized with rSaEsxA and rSaEsxB had significantly improved survival rates when challenged with S. aureus compared with the controls. These findings indicate that SaEsxA and SaEsxB are two promising Th1 and Th17 candidate antigens, which could be developed into multivalent and serotype-independent vaccines against S. aureus infection.
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Signature gene expression reveals novel clues to the molecular mechanisms of dimorphic transition in Penicillium marneffei.
PLoS Genet.
PUBLISHED: 10-01-2014
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Systemic dimorphic fungi cause more than one million new infections each year, ranking them among the significant public health challenges currently encountered. Penicillium marneffei is a systemic dimorphic fungus endemic to Southeast Asia. The temperature-dependent dimorphic phase transition between mycelium and yeast is considered crucial for the pathogenicity and transmission of P. marneffei, but the underlying mechanisms are still poorly understood. Here, we re-sequenced P. marneffei strain PM1 using multiple sequencing platforms and assembled the genome using hybrid genome assembly. We determined gene expression levels using RNA sequencing at the mycelial and yeast phases of P. marneffei, as well as during phase transition. We classified 2,718 genes with variable expression across conditions into 14 distinct groups, each marked by a signature expression pattern implicated at a certain stage in the dimorphic life cycle. Genes with the same expression patterns tend to be clustered together on the genome, suggesting orchestrated regulations of the transcriptional activities of neighboring genes. Using qRT-PCR, we validated expression levels of all genes in one of clusters highly expressed during the yeast-to-mycelium transition. These included madsA, a gene encoding MADS-box transcription factor whose gene family is exclusively expanded in P. marneffei. Over-expression of madsA drove P. marneffei to undergo mycelial growth at 37°C, a condition that restricts the wild-type in the yeast phase. Furthermore, analyses of signature expression patterns suggested diverse roles of secreted proteins at different developmental stages and the potential importance of non-coding RNAs in mycelium-to-yeast transition. We also showed that RNA structural transition in response to temperature changes may be related to the control of thermal dimorphism. Together, our findings have revealed multiple molecular mechanisms that may underlie the dimorphic transition in P. marneffei, providing a powerful foundation for identifying molecular targets for mechanism-based interventions.
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Characterization of the antigenicity of Cpl1 protein, a surface protein of Cryptococcus neoformans var. neoformans.
Mycologia
PUBLISHED: 09-28-2014
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Cryptococcus neoformans var. neoformans is an important fungal pathogen. The capsule is a well established virulence factor and a target site for diagnostic tests. The CPL1 gene is required for capsular formation and virulence. The protein product Cpl1 has been proposed to be a secreted protein, but the characteristics of the protein have not been reported. Here we sought to characterize Cpl1. Phylogenetic analysis showed that the Cpl1 of C. neoformans var. neoformans and the Cpl1 orthologs identified in C. neoformans var. grubii and C. gattii formed a distinct cluster among related fungi; while the putative ortholog found in Trichosporon asahii was distantly related to the Cryptococcus cluster. We expressed Cpl1 abundantly as a secreted His-tagged protein in Pichia pastors. The protein was used to immunize guinea pigs and rabbits for high titer mono-specific polyclonal antibody that was shown to be highly specific against the cell wall of C. neoformans var. neoformans and did not cross react with C. gattii, T. asahii, Aspergillus spp., Candida spp. and Penicillium spp. Using the anti-Cpl1 antibody, we detected Cpl1 protein in the fresh culture supernatant of C. neoformans var. neoformans and we showed by immunostaining that the Cpl1 protein was located on the surface. The Cpl1 protein is a specific surface protein of C. neoformans var. neoformans.
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Fatal anti-aquaporin-4 seropositive neuromyelitis optica spectrum disorder in tuberculosis.
BMC Infect. Dis.
PUBLISHED: 08-28-2014
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Neuromyelitis optica (NMO) is an autoimmune inflammatory condition of the central nervous system that is characterized by circulating anti-aquaporin-4 antibodies, transverse myelitis and optic neuritis. NMO spectrum disorders are rarely reported in patients with active pulmonary tuberculosis (TB). We report a fatal case of anti-aquaporin-4 antibody positive NMO spectrum disorder in a patient who was receiving treatment for pulmonary tuberculosis.
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Vaccine-Elicited CD8+ T Cells Cure Mesothelioma by Overcoming Tumor-Induced Immunosuppressive Environment.
Cancer Res.
PUBLISHED: 08-14-2014
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Eradicating malignant tumors by vaccine-elicited host immunity remains a major medical challenge. To date, correlates of immune protection remain unknown for malignant mesothelioma. In this study, we demonstrated that antigen-specific CD8(+) T-cell immune response correlates with the elimination of malignant mesothelioma by a model PD-1-based DNA vaccine. Unlike the nonprotective tumor antigen WT1-based DNA vaccines, the model vaccine showed complete and long-lasting protection against lethal mesothelioma challenge in immunocompetent BALB/c mice. Furthermore, it remained highly immunogenic in tumor-bearing animals and led to therapeutic cure of preexisting mesothelioma. T-cell depletion and adoptive transfer experiments revealed that vaccine-elicited CD8(+) T cells conferred to the protective efficacy in a dose-dependent way. Also, these CD8(+) T cells functioned by releasing inflammatory IFN? and TNF? in the vicinity of target cells as well as by initiating TRAIL-directed tumor cell apoptosis. Importantly, repeated DNA vaccinations, a major advantage over live-vectored vaccines with issues of preexisting immunity, achieve an active functional state, not only preventing the rise of exhausted PD-1(+) and Tim-3(+) CD8(+) T cells but also suppressing tumor-induced myeloid-derived suppressive cells and Treg cells, with the frequency of antigen-specific CD8(+) T cells inversely correlating with tumor mass. Our results provide new insights into quantitative and qualitative requirements of vaccine-elicited functional CD8(+) T cells in cancer prevention and immunotherapy. Cancer Res; 74(21); 6010-21. ©2014 AACR.
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Fatal systemic necrotizing infections associated with a novel paramyxovirus, anaconda paramyxovirus, in green anaconda juveniles.
J. Clin. Microbiol.
PUBLISHED: 07-30-2014
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Beginning in July 2011, 31 green anaconda (Eunectes murinus) juveniles from an oceanarium in Hong Kong died over a 12-month period. Necropsy revealed at least two of the following features in 23 necropsies: dermatitis, severe pan-nephritis, and/or severe systemic multiorgan necrotizing inflammation. Histopathological examination revealed severe necrotizing inflammation in various organs, most prominently the kidneys. Electron microscopic examination of primary tissues revealed intralesional accumulations of viral nucleocapsids with diameters of 10 to 14 nm, typical of paramyxoviruses. Reverse transcription (RT)-PCR results were positive for paramyxovirus (viral loads of 2.33 × 10(4) to 1.05 × 10(8) copies/mg tissue) in specimens from anaconda juveniles that died but negative in specimens from the two anaconda juveniles and anaconda mother that survived. None of the other snakes in the park was moribund, and RT-PCR results for surveillance samples collected from other snakes were negative. The virus was isolated from BHK21 cells, causing cytopathic effects with syncytial formation. The virus could also replicate in 25 of 27 cell lines of various origins, in line with its capability for infecting various organs. Electron microscopy with cell culture material revealed enveloped virus with the typical "herringbone" appearance of helical nucleocapsids in paramyxoviruses. Complete genome sequencing of five isolates confirmed that the infections originated from the same clone. Comparative genomic and phylogenetic analyses and mRNA editing experiments revealed a novel paramyxovirus in the genus Ferlavirus, named anaconda paramyxovirus, with a typical Ferlavirus genomic organization of 3'-N-U-P/V/I-M-F-HN-L-5'. Epidemiological and genomic analyses suggested that the anaconda juveniles acquired the virus perinatally from the anaconda mother rather than from other reptiles in the park, with subsequent interanaconda juvenile transmission.
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Immunogenicity of intradermal trivalent influenza vaccine with topical imiquimod: a double blind randomized controlled trial.
Clin. Infect. Dis.
PUBLISHED: 07-21-2014
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Imiquimod, a synthetic Toll-like receptor 7 agonist enhanced immunogenicity of influenza vaccine in a mouse model. We hypothesized that topical imiquimod before intradermal influenza vaccination (TIV) would produce similar effect in human.
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The biosynthetic pathway for a thousand-year-old natural food colorant and citrinin in Penicillium marneffei.
Sci Rep
PUBLISHED: 07-10-2014
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Monascorubrin and its derivatives are polyketides used as natural colorants for a wide range of food for more than one thousand years. Since the biosynthetic pathway for this ancient chemical compound is unknown and genome sequence unavailable for any Monascus species, monascorubrin production has relied on extraction from fungal cultures of Monascus species. In vitro synthesis and genetic manipulation are not possible. Here we report the polyketide gene cluster and pathway for monascorubrin biosynthesis in Penicillium marneffei, a diffusible red pigment-producing, thermal dimorphic fungus, taking advantage of available genome sequence and faster growth rate than Monascus species. We also documented that the red pigment of P. marneffei is a mixture of more than 16 chemical compounds, which are amino acid conjugates of monascorubrin and rubropunctatin, and showed that this polyketide gene cluster and pathway are also responsible for biosynthesis of ankaflavin and citrinin, a mycotoxin with nephrotoxic activity in mammals. The present study on elucidation of the biosynthetic pathway of monascorubrin is a proof-of-the-concept study that serves as a cornerstone for future studies on monascorubrin biosynthesis pathway dissection in Monascus species.
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The Role of Hibiscus sabdariffa L. (Roselle) in Maintenance of Ex Vivo Murine Bone Marrow-Derived Hematopoietic Stem Cells.
ScientificWorldJournal
PUBLISHED: 06-11-2014
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Hematopoietic stem cells- (HSCs-) based therapy requires ex vivo expansion of HSCs prior to therapeutic use. However, ex vivo culture was reported to promote excessive production of reactive oxygen species (ROS), exposing HSCs to oxidative damage. Efforts to overcome this limitation include the use of antioxidants. In this study, the role of Hibiscus sabdariffa L. (Roselle) in maintenance of cultured murine bone marrow-derived HSCs was investigated. Aqueous extract of Roselle was added at varying concentrations (0-1000?ng/mL) for 24 hours to the freshly isolated murine bone marrow cells (BMCs) cultures. Effects of Roselle on cell viability, reactive oxygen species (ROS) production, glutathione (GSH) level, superoxide dismutase (SOD) activity, and DNA damage were investigated. Roselle enhanced the survival (P < 0.05) of BMCs at 500 and 1000?ng/mL, increased survival of Sca-1(+) cells (HSCs) at 500?ng/mL, and maintained HSCs phenotype as shown from nonremarkable changes of surface marker antigen (Sca-1) expression in all experimental groups. Roselle increased (P < 0.05) the GSH level and SOD activity but the level of reactive oxygen species (ROS) was unaffected. Moreover, Roselle showed significant cellular genoprotective potency against H2O2-induced DNA damage. Conclusively, Roselle shows novel property as potential supplement and genoprotectant against oxidative damage to cultured HSCs.
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Decolonization of gastrointestinal carriage of vancomycin-resistant Enterococcus faecium: case series and review of literature.
BMC Infect. Dis.
PUBLISHED: 06-06-2014
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Prolonged asymptomatic carriage of vancomycin-resistant enterococci (VRE) in the gastrointestinal tract and the lack of effective decolonization regimen perpetuate the endemicity of VRE in the healthcare settings.
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Chickens host diverse picornaviruses originated from potential interspecies transmission with recombination.
J. Gen. Virol.
PUBLISHED: 06-06-2014
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While chickens are an important reservoir for emerging pathogens such as avian influenza viruses, little is known about the diversity of picornaviruses in poultry. We discovered a previously unknown diversity of picornaviruses in chickens in Hong Kong. Picornaviruses were detected in 87 cloacal and 7 tracheal samples from 93 of 900 chickens by reverse transcription-PCR, with their partial 3D(pol) gene sequences forming five distinct clades (I to V) among known picornaviruses. Analysis of eight genomes from different clades revealed seven different picornaviruses, including six novel picornavirus species (ChPV1 from clade I, ChPV2 and ChPV3 from clade II, ChPV4 and ChPV5 from clade III, ChGV1 from clade IV) and one existing species (Avian encephalomyelitis virus from clade V). The six novel chicken picornavirus genomes exhibited distinct phylogenetic positions and genome features different from related picornaviruses, supporting their classification as separate species. Moreover, ChPV1 may potentially belong to a novel genus, with low sequence homologies to related picornaviruses, especially in the P1 and P2 regions, including the predicted L and 2A proteins. Nevertheless, these novel picornaviruses were most closely related to picornaviruses of other avian species (ChPV1 related to Passerivirus A, ChPV2 and ChPV3 to Avisivirus A and Duck hepatitis A virus, ChPV4 and ChPV5 to Melegrivirus A, ChGV1 to Gallivirus A). Furthermore, ChPV5 represented a potential recombinant picornavirus, with its P2 and P3 regions possibly originating from Melegrivirus A. Chickens are an important reservoir for diverse picornaviruses that may cross avian species barriers through mutation or recombination.
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New hepatitis E virus genotype in camels, the Middle East.
Emerging Infect. Dis.
PUBLISHED: 05-27-2014
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In a molecular epidemiology study of hepatitis E virus (HEV) in dromedaries in Dubai, United Arab Emirates, HEV was detected in fecal samples from 3 camels. Complete genome sequencing of 2 strains showed >20% overall nucleotide difference to known HEVs. Comparative genomic and phylogenetic analyses revealed a previously unrecognized HEV genotype.
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Epidemiology of human parechovirus, Aichi virus and salivirus in fecal samples from hospitalized children with gastroenteritis in Hong Kong.
Virol. J.
PUBLISHED: 05-22-2014
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Emerging human picornaviruses, including human parechovirus (HPeV), Aichi virus (AiV) and salivirus (SalV) were found to be associated with gastroenteritis, but their roles in enteric infections are not fully understood. In addition, no report on the circulation of these viruses in Hong Kong is available. The objective of this study was to investigate the prevalence and genetic diversity of HPeV, AiV and SalV in fecal samples from hospitalized children with gastroenteritis in Hong Kong.
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Bat origins of MERS-CoV supported by bat coronavirus HKU4 usage of human receptor CD26.
Cell Host Microbe
PUBLISHED: 05-14-2014
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The recently reported Middle East respiratory syndrome coronavirus (MERS-CoV) is phylogenetically closely related to the bat coronaviruses (BatCoVs) HKU4 and HKU5. However, the evolutionary pathway of MERS-CoV is still unclear. A receptor binding domain (RBD) in the MERS-CoV envelope-embedded spike protein specifically engages human CD26 (hCD26) to initiate viral entry. The high sequence identity in the viral spike protein prompted us to investigate if HKU4 and HKU5 can recognize hCD26 for cell entry. We found that HKU4-RBD, but not HKU5-RBD, binds to hCD26, and pseudotyped viruses embedding HKU4 spike can infect cells via hCD26 recognition. The structure of the HKU4-RBD/hCD26 complex revealed a hCD26-binding mode similar overall to that observed for MERS-RBD. HKU4-RBD, however, is less adapted to hCD26 than MERS-RBD, explaining its lower affinity for receptor binding. Our findings support a bat origin for MERS-CoV and indicate the need for surveillance of HKU4-related viruses in bats.
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Exceptionally potent neutralization of Middle East respiratory syndrome coronavirus by human monoclonal antibodies.
J. Virol.
PUBLISHED: 04-30-2014
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The recently discovered Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans, with high mortality. Specific, highly effective therapeutics and vaccines against the MERS-CoV are urgently needed to save human lives and address the pandemic concerns. We identified three human monoclonal antibodies (MAbs), m336, m337, and m338, targeting the receptor (CD26/DPP4) binding domain (RBD) of the MERS-CoV spike glycoprotein from a very large naïve-antibody library (containing ?10(11) antibodies). They bound with high affinity: equilibrium dissociation constants for the three MAbs were equal to 4.2, 9.3, and 15 nM, respectively, as measured by Biacore for Fabs binding to RBD. The avidity for IgG1 m336, m337, and m338 was even higher: 99, 820, and 560 pM, respectively. The antibodies bound to overlapping epitopes that overlap the receptor binding site on the RBD as suggested by competition experiments and further supported by site-directed mutagenesis of the RBD and a docking model of the m336-RBD complex. The highest-affinity MAb, m336, neutralized both pseudotyped and live MERS-CoV with exceptional potency, 50% neutralization at 0.005 and 0.07 ?g/ml, respectively, likely by competing with DPP4 for binding to the S glycoprotein. The exceptionally high neutralization activity of these antibodies and especially m336 suggests that they have great potential for prophylaxis and therapy of MERS-CoV infection in humans and as a tool for development of vaccine immunogens. The rapid identification (within several weeks) of potent MAbs suggests a possibility to use the new large antibody library and related methodology for a quick response to the public threat resulting from emerging coronaviruses. Importance: A novel human coronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV), was found to infect humans with a high mortality rate in 2012, just 1 decade after the appearance of the first highly pathogenic coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV). There are no effective therapeutics available. It is highly desirable to find an approach for rapidly developing potent therapeutics against MERS-CoV, which not only can be implemented for MERS treatment but also can help to develop a platform strategy to combat future emerging coronaviruses. We report here the identification of human monoclonal antibodies (MAbs) from a large nonimmune antibody library that target MERS-CoV. One of the antibodies, m336, neutralized the virus with exceptional potency. It therefore may have great potential as a candidate therapeutic and as a reagent to facilitate the development of vaccines against MERS-CoV.
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Potent neutralization of MERS-CoV by human neutralizing monoclonal antibodies to the viral spike glycoprotein.
Sci Transl Med
PUBLISHED: 04-28-2014
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The recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and fatal acute respiratory illness in humans. However, no prophylactic and therapeutic agents specifically against MERS-CoV are currently available. Entry of MERS-CoV into target cells depends on binding of the receptor binding domain (RBD) of the viral envelope spike glycoprotein to the cellular receptor dipeptidyl peptidase 4 (DPP4). We report the isolation and characterization of two potent human RBD-specific neutralizing monoclonal antibodies (MERS-4 and MERS-27) derived from single-chain variable region fragments of a nonimmune human antibody library. MERS-4 and MERS-27 inhibited infection of both pseudotyped and live MERS-CoV with IC50 (half-maximal inhibitory concentration) at nanomolar concentrations. MERS-4 also showed inhibitory activity against syncytia formation mediated by interaction between MERS-CoV spike glycoprotein and DPP4. Combination of MERS-4 and MERS-27 demonstrated a synergistic effect in neutralization against pseudotyped MERS-CoV. Biochemical analysis indicated that MERS-4 and MERS-27 blocked RBD interaction with DPP4 on the cell surface. MERS-4, in particular, bound soluble RBD with an about 45-fold higher affinity than DPP4. Mutagenesis analysis suggested that MERS-4 and MERS-27 recognized distinct regions in RBD. These results suggest that MERS-4 and MERS-27 are RBD-specific potent inhibitors and could serve as promising candidates for prophylactic and therapeutic interventions against MERS-CoV infection.
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Proactive infection control measures to prevent nosocomial transmission of vancomycin-resistant enterococci in Hong Kong.
J. Formos. Med. Assoc.
PUBLISHED: 03-31-2014
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The study describes a proactive infection control approach to prevent nosocomial transmission of vancomycin-resistant enterococci (VRE) and tests if this approach is effective for controlling multiple-drug resistant organisms in a nonendemic setting.
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Molecular diagnosis in clinical parasitology: When and why?
Exp. Biol. Med. (Maywood)
PUBLISHED: 03-25-2014
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Microscopic detection and morphological identification of parasites from clinical specimens are the gold standards for the laboratory diagnosis of parasitic infections. The limitations of such diagnostic assays include insufficient sensitivity and operator dependence. Immunoassays for parasitic antigens are not available for most parasitic infections and have not significantly improved the sensitivity of laboratory detection. Advances in molecular detection by nucleic acid amplification may improve the detection in asymptomatic infections with low parasitic burden. Rapidly accumulating genomic data on parasites allow the design of polymerase chain reaction (PCR) primers directed towards multi-copy gene targets, such as the ribosomal and mitochondrial genes, which further improve the sensitivity. Parasitic cell or its free circulating parasitic DNA can be shed from parasites into blood and excreta which may allow its detection without the whole parasite being present within the portion of clinical sample used for DNA extraction. Multiplex nucleic acid amplification technology allows the simultaneous detection of many parasitic species within a single clinical specimen. In addition to improved sensitivity, nucleic acid amplification with sequencing can help to differentiate different parasitic species at different stages with similar morphology, detect and speciate parasites from fixed histopathological sections and identify anti-parasitic drug resistance. The use of consensus primer and PCR sequencing may even help to identify novel parasitic species. The key limitation of molecular detection is the technological expertise and expense which are usually lacking in the field setting at highly endemic areas. However, such tests can be useful for screening important parasitic infections in asymptomatic patients, donors or recipients coming from endemic areas in the settings of transfusion service or tertiary institutions with transplantation service. Such tests can also be used for monitoring these recipients or highly immunosuppressed patients, so that early preemptive treatment can be given for reactivated parasitic infections while the parasitic burden is still low.
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Novel betacoronavirus in dromedaries of the Middle East, 2013.
Emerging Infect. Dis.
PUBLISHED: 03-25-2014
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In 2013, a novel betacoronavirus was identified in fecal samples from dromedaries in Dubai, United Arab Emirates. Antibodies against the recombinant nucleocapsid protein of the virus, which we named dromedary camel coronavirus (DcCoV) UAE-HKU23, were detected in 52% of 59 dromedary serum samples tested. In an analysis of 3 complete DcCoV UAE-HKU23 genomes, we identified the virus as a betacoronavirus in lineage A1. The DcCoV UAE-HKU23 genome has G+C contents; a general preference for G/C in the third position of codons; a cleavage site for spike protein; and a membrane protein of similar length to that of other betacoronavirus A1 members, to which DcCoV UAE-HKU23 is phylogenetically closely related. Along with this coronavirus, viruses of at least 8 other families have been found to infect camels. Because camels have a close association with humans, continuous surveillance should be conducted to understand the potential for virus emergence in camels and for virus transmission to humans.
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Emergence of scarlet fever Streptococcus pyogenes emm12 clones in Hong Kong is associated with toxin acquisition and multidrug resistance.
Nat. Genet.
PUBLISHED: 03-20-2014
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A scarlet fever outbreak began in mainland China and Hong Kong in 2011 (refs. 1-6). Macrolide- and tetracycline-resistant Streptococcus pyogenes emm12 isolates represent the majority of clinical cases. Recently, we identified two mobile genetic elements that were closely associated with emm12 outbreak isolates: the integrative and conjugative element ICE-emm12, encoding genes for tetracycline and macrolide resistance, and prophage ?HKU.vir, encoding the superantigens SSA and SpeC, as well as the DNase Spd1 (ref. 4). Here we sequenced the genomes of 141 emm12 isolates, including 132 isolated in Hong Kong between 2005 and 2011. We found that the introduction of several ICE-emm12 variants, ?HKU.vir and a new prophage, ?HKU.ssa, occurred in three distinct emm12 lineages late in the twentieth century. Acquisition of ssa and transposable elements encoding multidrug resistance genes triggered the expansion of scarlet fever-associated emm12 lineages in Hong Kong. The occurrence of multidrug-resistant ssa-harboring scarlet fever strains should prompt heightened surveillance within China and abroad for the dissemination of these mobile genetic elements.
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Viral lung infections: epidemiology, virology, clinical features, and management of avian influenza A(H7N9).
Curr Opin Pulm Med
PUBLISHED: 03-19-2014
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The avian influenza A(H7N9) virus has jumped species barrier and caused severe human infections. Here, we present the virological features relevant to clinical practice, and summarize the epidemiology, clinical findings, diagnosis, treatment, and preventive strategies of A(H7N9) infection.
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NF90 exerts antiviral activity through regulation of PKR phosphorylation and stress granules in infected cells.
J. Immunol.
PUBLISHED: 03-12-2014
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NF90 was shown to exhibit broad antiviral activity against several viruses, but detailed mechanisms remain unclear. In this study, we examined the molecular basis for the inhibitory effect of NF90 on virus replication mediated through protein kinase (PKR)-associated translational regulation. We first verified the interaction between NF90 and PKR in mammalian cells and showed that NF90 interacts with PKR through its C-terminal and that the interaction is independent of NF90 RNA-binding properties. We further showed that knockdown of NF90 resulted in significantly lower levels of PKR phosphorylation in response to dsRNA induction and influenza virus infection. We also showed that high concentrations of NF90 exhibit negative regulatory effects on PKR phosphorylation, presumably through competition for dsRNA via the C-terminal RNA-binding domain. PKR activation is essential for the formation of stress granules in response to dsRNA induction. Our results showed that NF90 is a component of stress granules. In NF90-knockdown cells, dsRNA treatment induced significantly lower levels of stress granules than in control cells. Further evidence for an NF90-PKR antiviral pathway was obtained using an NS1 mutated influenza A virus specifically attenuated in its ability to inhibit PKR activation. This mutant virus replicated indistinguishably from wild-type virus in NF90-knockdown cells, but not in scrambled control cells or Vero cells, indicating that NF90's antiviral function occurs through interaction with PKR. Taken together, these results reveal a yet-to-be defined host antiviral mechanism in which NF90 upregulation of PKR phosphorylation restricts virus infection.
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NMR-based metabolomic urinalysis: a rapid screening test for urinary tract infection.
Clin. Chim. Acta
PUBLISHED: 02-28-2014
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Urinary tract infection (UTI) is one of the most common bacterial infections in humans; however, there is no accurate and fast quantitative test to detect UTI. Dipstick urinalysis is semi-quantitative with a limited diagnostic accuracy, while urine culture is accurate but takes time. We described a quantitative biochemical method for the diagnosis of bacteriuria using a single marker.
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Unique reassortant of influenza A(H7N9) virus associated with severe disease emerging in Hong Kong.
J. Infect.
PUBLISHED: 02-19-2014
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Human infections caused by avian influenza virus A(H7N9) re-emerged in late 2013. We reported the first Hong Kong patient without risk factors for severe A(H7N9) disease.
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Middle east respiratory syndrome coronavirus 4a protein is a double-stranded RNA-binding protein that suppresses PACT-induced activation of RIG-I and MDA5 in the innate antiviral response.
J. Virol.
PUBLISHED: 02-12-2014
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Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes severe disease in human. MERS-CoV is closely related to bat coronaviruses HKU4 and HKU5. Evasion of the innate antiviral response might contribute significantly to MERS-CoV pathogenesis, but the mechanism is poorly understood. In this study, we characterized MERS-CoV 4a protein as a novel immunosuppressive factor that antagonizes type I interferon production. MERS-CoV 4a protein contains a double-stranded RNA-binding domain capable of interacting with poly(I · C). Expression of MERS-CoV 4a protein suppressed the interferon production induced by poly(I · C) or Sendai virus. RNA binding of MERS-CoV 4a protein was required for IFN antagonism, a property shared by 4a protein of bat coronavirus HKU5 but not by the counterpart in bat coronavirus HKU4. MERS-CoV 4a protein interacted with PACT in an RNA-dependent manner but not with RIG-I or MDA5. It inhibited PACT-induced activation of RIG-I and MDA5 but did not affect the activity of downstream effectors such as RIG-I, MDA5, MAVS, TBK1, and IRF3. Taken together, our findings suggest a new mechanism through which MERS-CoV employs a viral double-stranded RNA-binding protein to circumvent the innate antiviral response by perturbing the function of cellular double-stranded RNA-binding protein PACT. PACT targeting might be a common strategy used by different viruses, including Ebola virus and herpes simplex virus 1, to counteract innate immunity.
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Toll-like receptor 7 agonist imiquimod in combination with influenza vaccine expedites and augments humoral immune responses against influenza A(H1N1)pdm09 virus infection in BALB/c mice.
Clin. Vaccine Immunol.
PUBLISHED: 02-12-2014
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Toll-like receptors (TLRs) of the innate immune system are known targets for enhancing vaccine efficacy. We investigated whether imiquimod, a synthetic TLR7 agonist, can expedite the immune response against influenza virus infection when combined with influenza vaccine. BALB/c mice were immunized intraperitoneally with monovalent A(H1N1)pdm09 vaccine combined with imiquimod (VCI) prior to intranasal inoculation with a lethal dose of mouse-adapted A(H1N1)pdm09 virus. For mice immunized 3 days before infection, the survival rates were significantly higher in the VCI group (60%, mean survival time[MST], 11 days) than in the vaccine-alone (30%; MST, 8.8 days), imiquimod-alone (5%; MST, 8.4 days), and phosphate-buffered saline (PBS) (0%; MST, 6.2 days) groups (P < 0.01). In the VCI group, 45 and 35% of the mice survived even when they were infected 2 days or 1 day after immunization. Virus-specific serum IgM, IgG, and neutralizing antibodies appeared earlier with higher geometric mean titers in the VCI group than in the control groups. The pulmonary viral load was significantly lower at all time points postinfection in the VCI, vaccine-alone, and imiquimod-alone groups than in the PBS control group (P < 0.05). The protection induced by VCI was specific for A(H1N1)pdm09 virus but not for A(H5N1) virus. Since imiquimod combined with RNase-treated vaccine is as protective as imiquimod combined with untreated vaccine, mechanisms other than TLR7 may operate in expediting and augmenting immune protection. Moreover, increased gamma interferon mRNA expression and IgG isotype switching, which are markers of the Th1 response induced by imiquimod, were not apparent in our mouse model. The mechanisms of imiquimod-induced immune protection deserve further study.
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Productive replication of Middle East respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response.
Virology
PUBLISHED: 02-06-2014
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The Middle East respiratory syndrome coronavirus (MERS-CoV) closely resembled severe acute respiratory syndrome coronavirus (SARS-CoV) in disease manifestation as rapidly progressive acute pneumonia with multi-organ dysfunction. Using monocyte-derived-dendritic cells (Mo-DCs), we discovered fundamental discrepancies in the outcome of MERS-CoV- and SARS-CoV-infection. First, MERS-CoV productively infected Mo-DCs while SARS-CoV-infection was abortive. Second, MERS-CoV induced significantly higher levels of IFN-?, IP-10, IL-12, and RANTES expression than SARS-CoV. Third, MERS-CoV-infection induced higher surface expression of MHC class II (HLA-DR) and the co-stimulatory molecule CD86 than SARS-CoV-infection. Overall, our data suggests that the dendritic cell can serve as an important target of viral replication and a vehicle for dissemination. MERS-CoV-infection in DCs results in the production of a rich combination of cytokines and chemokines, and modulates innate immune response differently from that of SARS-CoV-infection. Our findings may help to explain the apparent discrepancy in the pathogenicity between MERS-CoV and SARS-CoV.
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Fatal empyema thoracis caused by Schizophyllum commune with cross-reactive cryptococcal antigenemia.
J. Clin. Microbiol.
PUBLISHED: 01-31-2014
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We report a fatal case of Schizophyllum commune empyema thoracis with cross-reactive cryptococcal antigenemia. In vitro testing confirmed the ability of the fungus to cause a positive cryptococcal antigen latex agglutination system (CALAS) test result. Such a result may lead to delay in diagnosis and treatment, as most strains of S. commune are resistant to fluconazole.
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Avian influenza A H7N9 virus induces severe pneumonia in mice without prior adaptation and responds to a combination of zanamivir and COX-2 inhibitor.
PLoS ONE
PUBLISHED: 01-01-2014
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Human infection caused by the avian influenza A H7N9 virus has a case-fatality rate of over 30%. Systematic study of the pathogenesis of avian H7N9 isolate and effective therapeutic strategies are needed.
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Proactive infection control measures to prevent nosocomial transmission of carbapenem-resistant Enterobacteriaceae in a non-endemic area.
Chin. Med. J.
PUBLISHED: 11-30-2013
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Identification of hospitalized carbapenem-resistant Enterobacteriaceae (CRE)-positive patient is important in preventing nosocomial transmission. The objective of this study was to illustrate the implementation of proactive infection control measures in preventing nosocomial transmission of CRE in a healthcare region of over 3200 beds in Hong Kong between October 1, 2010 and December 31, 2011.
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Discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in gammacoronavirus.
J. Virol.
PUBLISHED: 11-13-2013
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While gammacoronaviruses mainly comprise infectious bronchitis virus (IBV) and its closely related bird coronaviruses (CoVs), the only mammalian gammacoronavirus was discovered from a white beluga whale (beluga whale CoV [BWCoV] SW1) in 2008. In this study, we discovered a novel gammacoronavirus from fecal samples from three Indo-Pacific bottlenose dolphins (Tursiops aduncus), which we named bottlenose dolphin CoV (BdCoV) HKU22. All the three BdCoV HKU22-positive samples were collected on the same date, suggesting a cluster of infection, with viral loads of 1 × 10(3) to 1 × 10(5) copies per ml. Clearance of virus was associated with a specific antibody response against the nucleocapsid of BdCoV HKU22. Complete genome sequencing and comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 have similar genome characteristics and structures. Their genome size is about 32,000 nucleotides, the largest among all CoVs, as a result of multiple unique open reading frames (NS5a, NS5b, NS5c, NS6, NS7, NS8, NS9, and NS10) between their membrane (M) and nucleocapsid (N) protein genes. Although comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 should belong to the same species, a major difference was observed in the proteins encoded by their spike (S) genes, which showed only 74.3 to 74.7% amino acid identities. The high ratios of the number of synonymous substitutions per synonymous site (Ks) to the number of nonsynonymous substitutions per nonsynonymous site (Ka) in multiple regions of the genome, especially the S gene (Ka/Ks ratio, 2.5), indicated that BdCoV HKU22 may be evolving rapidly, supporting a recent transmission event to the bottlenose dolphins. We propose a distinct species, Cetacean coronavirus, in Gammacoronavirus, to include BdCoV HKU22 and BWCoV SW1, whereas IBV and its closely related bird CoVs represent another species, Avian coronavirus, in Gammacoronavirus.
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Solving the mystery of H7N9 by crystal balls.
Cell Res.
PUBLISHED: 11-12-2013
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How the novel influenza H7N9 virus crossed species barrier from avian to human is intriguing. Extrapolation from previous studies on H5N1 can be misleading as illustrated by crystallographic studies on the H7 hemagglutinin with G226L substitution; crystal structure of the neuraminidase N9 showed that R294K substitution interferes with binding to sialic acid or antiviral drugs and reduces viral fitness.
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An Animal Model of MERS Produced by Infection of Rhesus Macaques With MERS Coronavirus.
J. Infect. Dis.
PUBLISHED: 11-11-2013
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In 2012, a novel coronavirus (CoV) associated with severe respiratory disease, Middle East respiratory syndrome (MERS-CoV; previously known as human coronavirus-Erasmus Medical Center or hCoV-EMC), emerged in the Arabian Peninsula. To date, 114 human cases of MERS-CoV have been reported, with 54 fatalities. Animal models for MERS-CoV infection of humans are needed to elucidate MERS pathogenesis and to develop vaccines and antivirals. In this study, we developed rhesus macaques as a model for MERS-CoV using intratracheal inoculation. The infected monkeys showed clinical signs of disease, virus replication, histological lesions, and neutralizing antibody production, indicating that this monkey model is suitable for studies of MERS-CoV infection.
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First Report of Brain Abscess Caused by a Satelliting Phenotypic Variant of Helcococcus kunzii.
J. Clin. Microbiol.
PUBLISHED: 10-30-2013
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Helcococcus kunzii was isolated from a brain abscess in a diabetic patient with cholesteatoma and demonstrated satellitism around Staphylococcus aureus in culture. This is the first reported case of severe central nervous system infection due to H. kunzii and the first description of a satelliting phenotypic variant of this organism.
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Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry for identification of clinically significant bacteria that are difficult to identify in clinical laboratories.
J. Clin. Pathol.
PUBLISHED: 10-18-2013
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Although the revolutionary matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has been evaluated for identification of various groups of bacteria, its application in bacteria that are difficult-to-identify by phenotypic tests has been less well studied. We aim to evaluate the usefulness of MALDI-TOF MS for identification of difficult-to-identify bacterial isolates.
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Antigenicity and transmissibility of a novel clade 2.3.2.1 avian influenza H5N1 virus.
J. Gen. Virol.
PUBLISHED: 09-28-2013
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A genetic variant of the H5N1 influenza virus, termed subclade 2.3.2.1, was first identified in Bulgaria in 2010 and has subsequently been found in Vietnam and Laos. Several cases of human infections with this virus have been identified. Thus, it is important to understand the antigenic properties and transmissibility of this variant. Our results showed that, although it is phylogenetically closely related to other previously characterized clade 2.3 viruses, this novel 2.3.2.1 variant exhibited distinct antigenic properties and showed little cross-reactivity to sera raised against other H5N1 viruses. Like other H5N1 viruses, this variant bound preferentially to avian-type receptors, but contained substitutions at positions 190 and 158 of the haemagglutinin (HA) protein that have been postulated to facilitate HA binding to human-type receptors and to enhance viral transmissibility among mammals, respectively. However, this virus did not appear to have acquired the capacity for airborne transmission between ferrets. These findings highlight the challenges in selecting vaccine candidates for H5N1 influenza because these viruses continue to evolve rapidly in the field. It is important to note that some variants have obtained mutations that may gain transmissibility between model animals, and close surveillance of H5N1 viruses in poultry is warranted.
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Delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel Middle East respiratory syndrome coronavirus: implications for pathogenesis and treatment.
J. Gen. Virol.
PUBLISHED: 09-28-2013
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The high mortality associated with the novel Middle East respiratory syndrome coronavirus (MERS-CoV) has raised questions about the possible role of a cytokine storm in its pathogenesis. Although recent studies showed that MERS-CoV infection is associated with an attenuated IFN response, no induction of inflammatory cytokines was demonstrated during the early phase of infection. To study both early and late cytokine responses associated with MERS-CoV infection, we measured the mRNA levels of eight cytokine genes [TNF-?, IL-1?, IL-6, IL-8, IFN-?, monocyte chemotactic protein-1, transforming growth factor-? and IFN-?-induced protein (IP)-10] in cell lysates of polarized airway epithelial Calu-3 cells infected with MERS-CoV or severe acute respiratory syndrome (SARS)-CoV up to 30 h post-infection. Among the eight cytokine genes, IL-1?, IL-6 and IL-8 induced by MERS-CoV were markedly higher than those induced by SARS-CoV at 30 h, whilst TNF-?, IFN-? and IP-10 induced by SARS-CoV were markedly higher than those induced by MERS-CoV at 24 and 30 h in infected Calu-3 cells. The activation of IL-8 and attenuated IFN-? response by MERS-CoV were also confirmed by protein measurements in the culture supernatant when compared with SARS-CoV and Sendai virus. To further confirm the attenuated antiviral response, cytokine response was compared with human HCoV-229E in embryonal lung fibroblast HFL cells, which also revealed higher IFN-? and IP-10 levels induced by HCoV-229E than MERS-CoV at 24 and 30 h. Whilst our data supported recent findings that MERS-CoV elicits attenuated innate immunity, this represents the first report to demonstrate delayed proinflammatory cytokine induction by MERS-CoV. Our results provide insights into the pathogenesis and treatment of MERS-CoV infections.
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Complete genome sequence of a novel feline astrovirus from a domestic cat in Hong Kong.
Genome Announc
PUBLISHED: 09-28-2013
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We report the first complete genome sequence of a feline astrovirus (FAstV), FAstV2 strain 1637F, identified from a domestic cat. The genome is 6,779 nucleotides (nt) in length and consists of three overlapping open reading frames (ORF1a-ORF1b-ORF2). Sequence analysis suggests that FAstV2 represents a new FAstV genotype that is closely related to human astroviruses.
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Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus.
J. Infect.
PUBLISHED: 09-24-2013
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Middle East respiratory syndrome coronavirus (MERS-CoV) has emerged to cause fatal infections in patients in the Middle East and traveler-associated secondary cases in Europe and Africa. Person-to-person transmission is evident in outbreaks involving household and hospital contacts. Effective antivirals are urgently needed.
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Active Replication of Middle East Respiratory Syndrome Coronavirus Replication and Aberrant Induction of Inflammatory Cytokines and Chemokines in Human Macrophages: Implications for Pathogenesis.
J. Infect. Dis.
PUBLISHED: 09-24-2013
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Middle East respiratory syndrome coronavirus (MERS-CoV) infection caused severe pneumonia and multiorgan dysfunction and had a higher crude fatality rate (around 50% vs 10%) than SARS coronavirus (SARS-CoV) infection. To understand the pathogenesis, we studied viral replication, cytokine/chemokine response, and antigen presentation in MERS-CoV-infected human monocyte-derived macrophages (MDMs) versus SARS-CoV-infected MDMs. Only MERS-CoV can replicate in MDMs. Both viruses were unable to significantly stimulate the expression of antiviral cytokines (interferon ? [IFN-?] and IFN-?) but induced comparable levels of tumor necrosis factor ? and interleukin 6. Notably, MERS-CoV induced significantly higher expression levels of interleukin 12, IFN-?, and chemokines (IP-10/CXCL-10, MCP-1/CCL-2, MIP-1?/CCL-3, RANTES/CCL-5, and interleukin 8) than SARS-CoV. The expression of major histocompatibility complex class I and costimulatory molecules were significantly higher in MERS-CoV-infected MDMs than in SARS-CoV-infected cells. MERS-CoV replication was validated by immunostaining of infected MDMs and ex vivo lung tissue. We conclusively showed that MERS-CoV can establish a productive infection in human macrophages. The aberrant induction of inflammatory cytokines/chemokines could be important in the disease pathogenesis.
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A unique and conserved neutralization epitope in H5N1 influenza viruses identified by an antibody against the A/Goose/Guangdong/1/96 hemagglutinin.
J. Virol.
PUBLISHED: 09-18-2013
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Despite substantial efforts to control and contain H5N1 influenza viruses, bird flu viruses continue to spread and evolve. Neutralizing antibodies against conserved epitopes on the viral hemagglutinin (HA) could confer immunity to the diverse H5N1 virus strains and provide information for effective vaccine design. Here, we report the characterization of a broadly neutralizing murine monoclonal antibody, H5M9, to most H5N1 clades and subclades that was elicited by immunization with viral HA of A/Goose/Guangdong/1/96 (H5N1), the immediate precursor of the current dominant strains of H5N1 viruses. The crystal structures of the Fab fragment of H5M9 in complexes with H5 HAs of A/Vietnam/1203/2004 and A/Goose/Guangdong/1/96 reveal a conserved epitope in the HA1 vestigial esterase subdomain that is some distance from the receptor binding site and partially overlaps antigenic site C of H3 HA. Further epitope characterization by selection of escape mutants and epitope mapping by flow cytometry analysis of site-directed mutagenesis of HA with a yeast cell surface display identified four residues that are critical for H5M9 binding. D53, Y274, E83a, and N276 are all conserved in H5N1 HAs and are not in H5 epitopes identified by other mouse or human antibodies. Antibody H5M9 is effective in protection of H5N1 virus both prophylactically and therapeutically and appears to neutralize by blocking both virus receptor binding and postattachment steps. Thus, the H5M9 epitope identified here should provide valuable insights into H5N1 vaccine design and improvement, as well as antibody-based therapies for treatment of H5N1 infection.
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Advantages of using matrix-assisted laser desorption ionization-time of flight mass spectrometry as a rapid diagnostic tool for identification of yeasts and mycobacteria in the clinical microbiological laboratory.
J. Clin. Microbiol.
PUBLISHED: 09-18-2013
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Yeast and mycobacteria can cause infections in immunocompromised patients and normal hosts. The rapid identification of these organisms can significantly improve patient care. There has been an increasing number of studies on using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid yeast and mycobacterial identifications. However, studies on direct comparisons between the Bruker Biotyper and bioMérieux Vitek MS systems for the identification of yeast and mycobacteria have been limited. This study compared the performance of the two systems in their identification of 98 yeast and 102 mycobacteria isolates. Among the 98 yeast isolates, both systems generated species-level identifications in >70% of the specimens, of which Candida albicans was the most commonly cultured species. At a genus-level identification, the Biotyper system identified more isolates than the Vitek MS system for Candida (75/78 [96.2%]versus 68/78 [87.2%], respectively; P = 0.0426) and non-Candida yeasts (18/20 [90.0%]versus 7/20 [35.0%], respectively; P = 0.0008). For mycobacterial identification, the Biotyper system generated reliable identifications for 89 (87.3%) and 64 (62.8%) clinical isolates at the genus and species levels, respectively, from solid culture media, whereas the Vitek MS system did not generate any reliable identification. The MS method differentiated 12/21 clinical species, despite the fact that no differentiation between Mycobacterium abscessus and Mycobacterium chelonae was found by using 16S rRNA gene sequencing. In summary, the MALDI-TOF MS method provides short turnaround times and a standardized working protocol for the identification of yeast and mycobacteria. Our study demonstrates that MALDI-TOF MS is suitable as a first-line test for the identification of yeast and mycobacteria in clinical laboratories.
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The emergence of influenza A H7N9 in human beings 16 years after influenza A H5N1: a tale of two cities.
Lancet Infect Dis
PUBLISHED: 08-24-2013
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Infection with either influenza A H5N1 virus in 1997 or avian influenza A H7N9 virus in 2013 caused severe pneumonia that did not respond to typical or atypical antimicrobial treatment, and resulted in high mortality. Both viruses are reassortants with internal genes derived from avian influenza A H9N2 viruses that circulate in Asian poultry. Both viruses have genetic markers of mammalian adaptation in their haemagglutinin and polymerase PB2 subunits, which enhanced binding to human-type receptors and improved replication in mammals, respectively. Hong Kong (affected by H5N1 in 1997) and Shanghai (affected by H7N9 in 2013) are two rapidly flourishing cosmopolitan megacities that were increasing in human population and poultry consumption before the outbreaks. Both cities are located along the avian migratory route at the Pearl River delta and Yangtze River delta. Whether the widespread use of the H5N1 vaccine in east Asia-with suboptimum biosecurity measures in live poultry markets and farms-predisposed to the emergence of H7N9 or other virus subtypes needs further investigation. Why H7N9 seems to be more readily transmitted from poultry to people than H5N1 is still unclear.
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Avian-Origin Influenza A(H7N9) Infection in Influenza A(H7N9)-Affected Areas of China: A Serological Study.
J. Infect. Dis.
PUBLISHED: 08-09-2013
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Serological surveillance conducted in areas of an outbreak of influenza A(H7N9) infection in China found no seropositivity for antibodies specific for avian-origin influenza A(H7N9) among 1129 individuals of the general population, whereas >6% of 396 poultry workers were positive (on the basis of a hemagglutination inhibition titer of ? 80) for this subtype, confirming that infected poultry is the principal source of human infections and that subclinical infections are possible. Fourteen days after symptom onset, elevated levels of antibodies to A(H7N9) were found in 65.8% of patients (25/38) who survived but in only 28.6% of those (2/7) who died, suggesting that the presence of antibodies may improve clinical outcome in infected patients.
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Identification and characterization of a novel paramyxovirus, porcine parainfluenza virus 1, from deceased pigs.
J. Gen. Virol.
PUBLISHED: 08-05-2013
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We describe the discovery and characterization of a novel paramyxovirus, porcine parainfluenza virus 1 (PPIV-1), from swine. The virus was detected in 12 (3.1?%) of 386 nasopharyngeal and two (0.7?%) of 303 rectal swab samples from 386 deceased pigs by reverse transcription-PCR, with viral loads of up to 10(6) copies ml(-1). Complete genome sequencing and phylogenetic analysis showed that PPIV-1 represented a novel paramyxovirus within the genus Respirovirus, being most closely related to human parainfluenza virus 1 (HPIV-1) and Sendai virus (SeV). In contrast to HPIV-1, PPIV-1 possessed a mRNA editing function in the phosphoprotein gene. Moreover, PPIV-1 was unique among respiroviruses in having two G residues instead of three to five G residues following the A6 run at the editing site. Nevertheless, PPIV-1, HPIV-1 and SeV share common genomic features and may belong to a separate group within the genus Respirovirus. The presence of PPIV-1 in mainly respiratory samples suggests a possible association with respiratory disease, similar to HPIV-1 and SeV.
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Identification of microRNA-like RNAs in mycelial and yeast phases of the thermal dimorphic fungus Penicillium marneffei.
PLoS Negl Trop Dis
PUBLISHED: 08-01-2013
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Penicillium marneffei is the most important thermal dimorphic fungus causing systemic mycosis in China and Southeast Asia. While miRNAs are increasingly recognized for their roles in post-transcriptional regulation of gene expression in animals and plants, miRNAs in fungi were less well studied and their potential roles in fungal dimorphism were largely unknown. Based on P. marneffei genome sequence, we hypothesize that miRNA-like RNAs (milRNAs) may be expressed in the dimorphic fungus.
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Proteome profiling of the dimorphic fungus Penicillium marneffei extracellular proteins and identification of glyceraldehyde-3-phosphate dehydrogenase as an important adhesion factor for conidial attachment.
FEBS J.
PUBLISHED: 07-29-2013
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Despite being the most important thermal dimorphic fungus causing systemic mycosis in Southeast Asia, the pathogenic mechanisms of Penicillium marneffei remain largely unknown. By comparing the extracellular proteomes of P. marneffei in mycelial and yeast phases, we identified 12 differentially expressed proteins among which glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat shock protein 60 (HSP60) were found to be upregulated in mycelial and yeast phases respectively. Based on previous findings in other pathogens, we hypothesized that these two extracellular proteins may be involved in adherence during P. marneffei-host interaction. Using inhibition assays with recombinant GAPDH (rGAPDH) proteins and anti-rGAPDH sera, we demonstrated that adhesion of P. marneffei conidia to fibronectin and laminin was inhibited by rGAPDH or rabbit anti-rGAPDH serum in a dose-dependent manner. Similarly, a dose-dependent inhibition of conidial adherence to A549 pneumocytes by rGAPDH or rabbit anti-rGAPDH serum was observed, suggesting that P. marneffei GAPDH can mediate binding of conidia to human extracellular matrix proteins and pneumocytes. However, HSP60 did not exhibit similar inhibition on conidia adherence, and neither GAPDH norHSP60 exhibited inhibition on adherence to J774 or THP-1 macrophage cell lines. This report demonstrates GAPDH as an adherence factor in P. marneffei by mediating conidia adherence to host bronchoalveolar epithelium during the early establishment phase of infection.
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Recombinant coxsackievirus A2 and deaths of children, Hong Kong, 2012.
Emerging Infect. Dis.
PUBLISHED: 07-24-2013
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A natural recombinant of coxsackievirus A2 was found in 4 children with respiratory symptoms in Hong Kong, China, during the summer of 2012. Two of these children died. Vigilant monitoring of this emerging recombinant enterovirus is needed to prevent its transmission to other regions.
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Unraveling the molecular basis of temperature-dependent genetic regulation in Penicillium marneffei.
Eukaryotic Cell
PUBLISHED: 07-12-2013
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Penicillium marneffei is an opportunistic fungal pathogen endemic in Southeast Asia, causing lethal systemic infections in immunocompromised patients. P. marneffei grows in a mycelial form at the ambient temperature of 25°C and transitions to a yeast form at 37°C. The ability to alternate between the mycelial and yeast forms at different temperatures, namely, thermal dimorphism, has long been considered critical for the pathogenicity of P. marneffei, yet the underlying genetic mechanisms remain elusive. Here we employed high-throughput sequencing to unravel global transcriptional profiles of P. marneffei PM1 grown at 25 and 37°C. Among ?11,000 protein-coding genes, 1,447 were overexpressed and 1,414 were underexpressed at 37°C. Counterintuitively, heat-responsive genes, predicted in P. marneffei through sequence comparison, did not tend to be overexpressed at 37°C. These results suggest that P. marneffei may take a distinct strategy of genetic regulation at the elevated temperature; the current knowledge concerning fungal heat response, based on studies of model fungal organisms, may not be applicable to P. marneffei. Our results further showed that the tandem repeat sequences (TRSs) are overrepresented in coding regions of P. marneffei genes, and TRS-containing genes tend to be overexpressed at 37°C. Furthermore, genomic sequences and expression data were integrated to characterize gene clusters, multigene families, and species-specific genes of P. marneffei. In sum, we present an integrated analysis and a comprehensive resource toward a better understanding of temperature-dependent genetic regulation in P. marneffei.
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Mycobacterium abscessus bacteremia after receipt of intravenous infusate of cytokine-induced killer cell therapy for body beautification and health boosting.
Clin. Infect. Dis.
PUBLISHED: 07-03-2013
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We report the first series of Mycobacterium abscessus bacteremia after cytokine-induced killer cell therapy for body beautification and health boosting.
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Human enterovirus 71 epidemics: whats next?
Emerg Health Threats J
PUBLISHED: 07-01-2013
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Human enterovirus 71 (EV71) epidemics have affected various countries in the past 40 years. EV71 commonly causes hand, foot and mouth disease (HFMD) in children, but can result in neurological and cardiorespiratory complications in severe cases. Genotypic changes of EV71 have been observed in different places over time, with the emergence of novel genotypes or subgenotypes giving rise to serious outbreaks. Since the late 1990s, intra- and inter-typic recombination events in EV71 have been increasingly reported in the Asia-Pacific region. In particular, double-recombinant EV71 strains belonging to a novel genotype D have been predominant in mainland China and Hong Kong over the last decade, though co-circulating with a minority of other EV71 subgenotypes and coxsackie A viruses. Continuous surveillance and genome studies are important to detect potential novel mutants or recombinants in the near future. Rapid and sensitive molecular detection of EV71 is of paramount importance in anticipating and combating EV71 outbreaks.
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Matrix-assisted laser desorption ionisation-time of flight mass spectrometry for rapid identification of Laribacter hongkongensis.
J. Clin. Pathol.
PUBLISHED: 06-28-2013
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Laribacter hongkongensis is a Gram-negative, facultative anaerobic, motile, S-shaped, urease-positive bacillus associated with invasive infections in liver cirrhosis patients and community-acquired gastroenteritis. Most cases of L hongkongensis infections occur in eastern countries. Information is lacking on the usefulness of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria important in eastern countries. Using the Bruker database extended with 21 L hongkongensis reference strains, all 240 L hongkongensis isolates recovered from patients, fish, frogs and water were correctly identified, with 224 (93.3%) strains having top match scores ?2.0. Notably, the strain of Chromobacterium violaceum was not reliably identified although it is included in the database. MALDI-TOF MS is useful for the accurate routine identification of L hongkongensis after adding reference L hongkongensis main spectra to the database. The number of strains for each species in MALDI-TOF MS databases should be expanded to cover intraspecies variability.
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Management of an incident of failed sterilization of surgical instruments in a dental clinic in Hong Kong.
J. Formos. Med. Assoc.
PUBLISHED: 06-10-2013
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We describe an investigation of an incident of failed sterilization procedure in a dental clinic. We aim to illustrate the principles in performing such investigations and to highlight some of the important checkpoints in sterilization procedures.
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Comparison of pyrosequencing, Sanger sequencing, and melting curve analysis for detection of low-frequency macrolide-resistant mycoplasma pneumoniae quasispecies in respiratory specimens.
J. Clin. Microbiol.
PUBLISHED: 05-29-2013
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Macrolide-resistant Mycoplasma pneumoniae (MRMP) is emerging worldwide and has been associated with treatment failure. In this study, we used pyrosequencing to detect low-frequency MRMP quasispecies in respiratory specimens, and we compared the findings with those obtained by Sanger sequencing and SimpleProbe PCR coupled with a melting curve analysis (SimpleProbe PCR). Sanger sequencing, SimpleProbe PCR, and pyrosequencing were successfully performed for 96.7% (88/91), 96.7% (88/91), and 93.4% (85/91) of the M. pneumoniae-positive specimens, respectively. The A-to-G transition at position 2063 was the only mutation identified. Pyrosequencing identified A2063G MRMP quasispecies populations in 78.8% (67/88) of the specimens. Only 38.8% (26/67) of these specimens with the A2063G quasispecies detected by pyrosequencing were found to be A2063G quasispecies by Sanger sequencing or SimpleProbe PCR. The specimens that could be detected by SimpleProbe PCR and Sanger sequencing had higher frequencies of MRMP quasispecies (51% to 100%) than those that could not be detected by those two methods (1% to 44%). SimpleProbe PCR correctly categorized all specimens that were identified as wild type or mutant by Sanger sequencing. The clinical characteristics of the patients were not significantly different when they were grouped by the presence or absence of MRMP quasispecies, while patients with MRMP identified by Sanger sequencing more often required a switch from macrolides to an alternative M. pneumoniae-targeted therapy. The clinical significance of mutant quasispecies should be investigated further with larger patient populations and with specimens obtained before and after macrolide therapy.
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Genetic characterization of Betacoronavirus lineage C viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus HKU5 in Japanese pipistrelle: implications for the origin of the novel Middle East respiratory sy
J. Virol.
PUBLISHED: 05-29-2013
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While the novel Middle East respiratory syndrome coronavirus (MERS-CoV) is closely related to Tylonycteris bat CoV HKU4 (Ty-BatCoV HKU4) and Pipistrellus bat CoV HKU5 (Pi-BatCoV HKU5) in bats from Hong Kong, and other potential lineage C betacoronaviruses in bats from Africa, Europe, and America, its animal origin remains obscure. To better understand the role of bats in its origin, we examined the molecular epidemiology and evolution of lineage C betacoronaviruses among bats. Ty-BatCoV HKU4 and Pi-BatCoV HKU5 were detected in 29% and 25% of alimentary samples from lesser bamboo bat (Tylonycteris pachypus) and Japanese pipistrelle (Pipistrellus abramus), respectively. Sequencing of their RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes revealed that MERS-CoV is more closely related to Pi-BatCoV HKU5 in RdRp (92.1% to 92.3% amino acid [aa] identity) but is more closely related to Ty-BatCoV HKU4 in S (66.8% to 67.4% aa identity) and N (71.9% to 72.3% aa identity). Although both viruses were under purifying selection, the S of Pi-BatCoV HKU5 displayed marked sequence polymorphisms and more positively selected sites than that of Ty-BatCoV HKU4, suggesting that Pi-BatCoV HKU5 may generate variants to occupy new ecological niches along with its host in diverse habitats. Molecular clock analysis showed that they diverged from a common ancestor with MERS-CoV at least several centuries ago. Although MERS-CoV may have diverged from potential lineage C betacoronaviruses in European bats more recently, these bat viruses were unlikely to be the direct ancestor of MERS-CoV. Intensive surveillance for lineage C betaCoVs in Pipistrellus and related bats with diverse habitats and other animals in the Middle East may fill the evolutionary gap.
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From SARS coronavirus to novel animal and human coronaviruses.
J Thorac Dis
PUBLISHED: 05-15-2013
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In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) caused one of the most devastating epidemics known to the developed world. There were two important lessons from this epidemic. Firstly, coronaviruses, in addition to influenza viruses, can cause severe and rapidly spreading human infections. Secondly, bats can serve as the origin and natural animal reservoir of deadly human viruses. Since then, researchers around the world, especially those in Asia where SARS-CoV was first identified, have turned their focus to find novel coronaviruses infecting humans, bats, and other animals. Two human coronaviruses, HCoV-HKU1 and HCoV-NL63, were identified shortly after the SARS-CoV epidemic as common causes of human respiratory tract infections. In 2012, a novel human coronavirus, now called Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in the Middle East to cause fatal human infections in three continents. MERS-CoV human infection is similar to SARS-CoV in having a high fatality rate and the ability to spread from person to person which resulted in secondary cases among close contacts including healthcare workers without travel history to the Middle East. Both viruses also have close relationships with bat coronaviruses. New cases of MERS-CoV infection in humans continue to occur with the origins of the virus still unknown in many cases. A multifaceted approach is necessary to control this evolving MERS-CoV outbreak. Source identification requires detailed epidemiological studies of the infected patients and enhanced surveillance of MERS-CoV or similar coronaviruses in humans and animals. Early diagnosis of infected patients and appropriate infection control measures will limit the spread in hospitals, while social distancing strategies may be necessary to control the outbreak in communities if it remained uncontrolled as in the SARS epidemic.
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Transmission of methicillin-resistant Staphylococcus aureus in the long term care facilities in Hong Kong.
BMC Infect. Dis.
PUBLISHED: 05-02-2013
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The relative contribution of long term care facilities (LTCFs) and hospitals in the transmission of methicillin-resistant Staphylococcus aureus (MRSA) is unknown.
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Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome.
Lancet
PUBLISHED: 04-25-2013
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Human infection with avian influenza A H7N9 virus emerged in eastern China in February, 2013, and has been associated with exposure to poultry. We report the clinical and microbiological features of patients infected with influenza A H7N9 virus and compare genomic features of the human virus with those of the virus in market poultry in Zhejiang, China.
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Interspecies transmission and emergence of novel viruses: lessons from bats and birds.
Trends Microbiol.
PUBLISHED: 03-22-2013
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As exemplified by coronaviruses and influenza viruses, bats and birds are natural reservoirs for providing viral genes during evolution of new virus species and viruses for interspecies transmission. These warm-blooded vertebrates display high species biodiversity, roosting and migratory behavior, and a unique adaptive immune system, which are favorable characteristics for asymptomatic shedding, dissemination, and mixing of different viruses for the generation of novel mutant, recombinant, or reassortant RNA viruses. The increased intrusion of humans into wildlife habitats and overcrowding of different wildlife species in wet markets and farms have also facilitated the interspecies transmission between different animal species.
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PD1-based DNA vaccine amplifies HIV-1 GAG-specific CD8+ T cells in mice.
J. Clin. Invest.
PUBLISHED: 02-22-2013
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Viral vector-based vaccines that induce protective CD8+ T cell immunity can prevent or control pathogenic SIV infections, but issues of preexisting immunity and safety have impeded their implementation in HIV-1. Here, we report the development of what we believe to be a novel antigen-targeting DNA vaccine strategy that exploits the binding of programmed death-1 (PD1) to its ligands expressed on dendritic cells (DCs) by fusing soluble PD1 with HIV-1 GAG p24 antigen. As compared with non-DC-targeting vaccines, intramuscular immunization via electroporation (EP) of the fusion DNA in mice elicited consistently high frequencies of GAG-specific, broadly reactive, polyfunctional, long-lived, and cytotoxic CD8+ T cells and robust anti-GAG antibody titers. Vaccination conferred remarkable protection against mucosal challenge with vaccinia GAG viruses. Soluble PD1-based vaccination potentiated CD8+ T cell responses by enhancing antigen binding and uptake in DCs and activation in the draining lymph node. It also increased IL-12-producing DCs and engaged antigen cross-presentation when compared with anti-DEC205 antibody-mediated DC targeting. The high frequency of durable and protective GAG-specific CD8+ T cell immunity induced by soluble PD1-based vaccination suggests that PD1-based DNA vaccines could potentially be used against HIV-1 and other pathogens.
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Genetic characterization of EV71 isolates from 2004 to 2010 reveals predominance and persistent circulation of the newly proposed genotype D and recent emergence of a distinct lineage of subgenotype C2 in Hong Kong.
Virol. J.
PUBLISHED: 02-07-2013
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Enterovirus 71 (EV71) is a common etiological agent of hand, foot and mouth disease (HFMD) in children. EV71 epidemics have been reported in Hong Kong in recent years, and yet the genetic information of EV71 strains circulating in our locality is limited. The objective of this study was to investigate the genetic evolution of these EV71 isolates in Hong Kong over a 7-year period.
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Reactive and infective dermatoses associated with adult-onset immunodeficiency due to anti-interferon-gamma autoantibody: Sweets syndrome and beyond.
Dermatology (Basel)
PUBLISHED: 01-14-2013
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Immunodeficiency due to anti-interferon-gamma autoantibody (anti-IFN-? autoAb) is an emerging adult-onset immunodeficiency syndrome predominantly found in Southeast Asians. It is associated with severe or disseminated infections caused by non-tuberculous mycobacteria (NTM) and other opportunistic pathogens. We describe 3 patients with anti-IFN-? autoAb who developed reactive and infective dermatoses, and thoroughly review the existing literature on dermatoses associated with the immunodeficiency syndrome. Case 1 developed Sweets syndrome associated with Mycobacterium chelonae lymphadenitis and penicilliosis. Case 2 suffered from multiple episodes of lobular panniculitis during recurrent infections by NTM, Penicillium marneffei and Burkholderia pseudomallei. Both cases responded to immunomodulating agents including corticosteroid and non-steroidal anti-inflammatory drugs. Case 3 had direct skin invasion by M. chelonae and responded to prolonged anti-mycobacterial therapy. A novel working algorithm is proposed for the diagnosis and treatment of these patients who may be encountered by the dermatologist and histopathologist in clinical practice.
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A Replicating Modified Vaccinia Tiantan Strain Expressing an Avian-Derived Influenza H5N1 Hemagglutinin Induce Broadly Neutralizing Antibodies and Cross-Clade Protective Immunity in Mice.
PLoS ONE
PUBLISHED: 01-01-2013
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To combat the possibility of a zoonotic H5N1 pandemic in a timely fashion, it is necessary to develop a vaccine that would confer protection against homologous and heterologous human H5N1 influenza viruses. Using a replicating modified vaccinia virus Tian Tan strain (MVTT) as a vaccine vector, we constructed MVTTHA-QH and MVTTHA-AH, which expresses the H5 gene of a goose-derived Qinghai strain A/Bar-headed Goose/Qinghai/1/2005 or human-derived Anhui Strain A/Anhui/1/2005. The immunogenicity profiles of both vaccine candidates were evaluated. Vaccination with MVTTHA-QH induced a significant level of neutralizing antibodies (Nabs) against a homologous strain and a wide range of H5N1 pseudoviruses (clades 1, 2.1, 2.2, 2.3.2, and 2.3.4). Neutralization tests (NT) and Haemagglutination inhibition (HI) antibodies inhibit the live autologous virus as well as a homologous A/Xingjiang/1/2006 and a heterologous A/Vietnam/1194/2004, representing two human isolates from clade 2.2 and clade 1, respectively. Importantly, mice vaccinated with intranasal MVTTHA-QH were completely protected from challenge with lethal dosages of A/Bar-headed Goose/Qinghai/1/2005 and the A/Viet Nam/1194/2004, respectively, but not control mice that received a mock MVTTS vaccine. However, MVTTHA-AH induced much lower levels of NT against its autologous strain. Our results suggest that it is feasible to use the H5 gene from A/Bar-headed Goose/Qinghai/1/2005 to construct an effective vaccine, when using MVTT as a vector, to prevent infections against homologous and genetically divergent human H5N1 influenza viruses.
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Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases.
PLoS ONE
PUBLISHED: 01-01-2013
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The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs).
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Natural occurrence and characterization of two internal ribosome entry site elements in a novel virus, canine picodicistrovirus, in the picornavirus-like superfamily.
J. Virol.
PUBLISHED: 12-28-2011
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Dicistroviridae and Picornaviridae are two phylogenetically related families of positive-sense single-stranded RNA viruses in the picornavirus-like superfamily with similar gene contents but different genome organizations and hosts. In a surveillance study involving 1,472 samples from 368 dogs over a 22-month period, we identified a novel picornavirus-like virus from 47 fecal and urine samples by the use of reverse transcription-PCR (RT-PCR). Sequencing and phylogenetic analysis of three complete genomes revealed that, although it seemed that the virus was most closely related to other picornaviruses, P1, P2, and P3 of the virus possessed very low amino acid identities of <30% to those of all other known picornaviruses and that the amino acid identities between the 3D(pol) and 2C of the virus and the RNA-dependent RNA polymerases and helicases of all other picornaviruses were <35%. Distinct from other picornaviruses, the genomes of the virus contain two putative internal ribosome entry sites (IRESs) and two open reading frames, encoding two polyprotein precursors (844 and 1,406 amino acids), separated by an intergenic region (IGR) of 588 bases. A dual-luciferase activity assay using DNA and RNA transfection revealed that both IRESs were functional. Quantitative RT-PCR showed that numbers of viral RNAs ranged from 7.55 × 10(6) to 1.26 × 10(9) copies/ml of urine and 1.82 × 10(6) to 4.97 × 10(10) copies/ml of fecal sample. This is the first report of the natural occurrence of two functional IRESs in nondicistroviruses. Based on our results, we have proposed a novel species, canine picodicistrovirus (CPDV), to describe this novel member of the picornavirus-like superfamily, which could represent a novel family of viruses.
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High performance weak donor-acceptor polymers in thin film transistors: effect of the acceptor on electronic properties, ambipolar conductivity, mobility, and thermal stability.
J. Am. Chem. Soc.
PUBLISHED: 12-05-2011
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We have studied the electronic, physical, and transistor properties of a family of donor-acceptor polymers consisting of diketopyrrolopyrrole (DPP) coupled with different accepting companion units in order to determine the effects of donor-acceptor interaction. Using the electronically neutral benzene (B), the weakly accepting benzothiadiazole (BT), and the strongly accepting benzobisthiadiazole (BBT), the accepting strength of the companion unit was systematically modulated. All polymers exhibited excellent transistor performance, with mobilities above 0.1 cm(2)V(-1)s(-1), even exceeding 1 cm(2)V(-1)s(-1) for one of the BBT-containing polymers. We find that the BBT is the strongest acceptor, enabling the BBT-containing polymers to be strongly ambipolar. The BBT moiety also strengthens interchain interactions, which provides higher thermal stability and performance for transistors with BBT-containing polymers as the active layer.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.