A novel bocavirus in canine liver

Background: Bocaviruses are classified as a genus within the Parvoviridae family of single-stranded DNA viruses and are pathogenic in some mammalian species. Two species have been previously reported in dogs, minute virus of canines (MVC), associated with neonatal diseases and fertility disorders; and Canine bocavirus (CBoV), associated with respiratory disease. Findings: In this study using deep sequencing of enriched viral particles from the liver of a dog with severe hemorrhagic gastroenteritis, necrotizing vasculitis, granulomatous lymphadenitis and anuric renal failure, we identified and characterized a novel bocavirus we named Canine bocavirus 3 (CnBoV3). The three major ORFs of CnBoV3 (NS1, NP1 and VP1) shared less than 60% aa identity with those of other bocaviruses qualifying it as a novel species based on ICTV criteria. Inverse PCR showed the presence of concatemerized or circular forms of the genome in liver. Conclusions: We genetically characterized a bocavirus in a dog liver that is highly distinct from prior canine bocaviruses found in respiratory and fecal samples. Its role in this animal’s complex disease remains to be determined. Keywords: Canine bocavirus 3; Episome; Coinfectio

Evaluation of oxacillin and cefoxitin disk diffusion and microbroth dilution methods for detecting mecA-mediated β-lactam resistance in contemporary Staphylococcus epidermidis isolates

Methicillin (β-lactam) resistance i

Enterovirus D68 outbreak detection through a syndromic disease epidemiology network

BACKGROUND: In 2014, enterovirus D68 (EV-D68) was responsible for an outbreak of severe respiratory illness in children, with 1,153 EV-D68 cases reported across 49 states. Despite this, there is no commercial assay for its detection in routine clinical care. BioFire® Syndromic Trends (Trend) is an epidemiological network that collects, in near real-time, deidentified. BioFire test results worldwide, including data from the BioFire® Respiratory Panel (RP). OBJECTIVES: Using the RP version 1.7 (which was not explicitly designed to differentiate EV-D68 from other picornaviruses), we formulate a model, Pathogen Extended Resolution (PER), to distinguish EV-D68 from other human rhinoviruses/enteroviruses (RV/EV) tested for in the panel. Using PER in conjunction with Trend, we survey for historical evidence of EVD68 positivity and demonstrate a method for prospective real-time outbreak monitoring within the network. STUDY DESIGN: PER incorporates real-time polymerase chain reaction metrics from the RPRV/EV assays. Six institutions in the United States and Europe contributed to the model creation, providing data from 1,619 samples spanning two years, confirmed by EV-D68 gold-standard molecular methods. We estimate outbreak periods by applying PER to over 600,000 historical Trend RP tests since 2014. Additionally, we used PER as a prospective monitoring tool during the 2018 outbreak. RESULTS: The final PER algorithm demonstrated an overall sensitivity and specificity of 87.1% and 86.1%, respectively, among the gold-standard dataset. During the 2018 outbreak monitoring period, PER alerted the research network of EV-D68 emergence in July. One of the first sites to experience a significant increase, Nationwide Children's Hospital, confirmed the outbreak and implemented EV-D68 testing at the institution in response. Applying PER to the historical Trend dataset to determine rates among RP tests, we find three potential outbreaks with predicted regional EV-D68 rates as high as 37% in 2014, 16% in 2016, and 29% in 2018. CONCLUSIONS: Using PER within the Trend network was shown to both accurately predict outbreaks of EV-D68 and to provide timely notifications of its circulation to participating clinical laboratories

Metagenomic next-generation sequencing of the 2014 Ebola Virus disease outbreak in the Democratic Republic of the Congo

We applied metagenomic next-generation sequencing (mNGS) to detect Zaire Ebola virus (EBOV) and other potential pathogens from whole-blood samples from 70 patients with suspected Ebola hemorrhagic fever during a 2014 outbreak in Boende, Democratic Republic of the Congo (DRC) and correlated these findings with clinical symptoms. Twenty of 31 patients (64.5%) tested in Kinshasa, DRC, were EBOV positive by quantitative reverse transcriptase PCR (qRT-PCR). Despite partial degradation of sample RNA during shipping and handling, mNGS followed by EBOV-specific capture probe enrichment in a U.S. genomics laboratory identified EBOV reads in 22 of 70 samples (31.4%) versus in 21 of 70 (30.0%) EBOV-positive samples by repeat qRT-PCR (overall concordance = 87.1%). Reads from Plasmodium falciparum (malaria) were detected in 21 patients, of which at least 9 (42.9%) were coinfected with EBOV. Other positive viral detections included hepatitis B virus (n = 2), human pegivirus 1 (n = 2), Epstein-Barr virus (n = 9), and Orungo virus (n = 1), a virus in the Reoviridae family. The patient with Orungo virus infection presented with an acute febrile illness and died rapidly from massive hemorrhage and dehydration. Although the patient's blood sample was negative by EBOV qRT-PCR testing, identification of viral reads by mNGS confirmed the presence of EBOV coinfection. In total, 9 new EBOV genomes (3 complete genomes, and an additional 6 ≥50% complete) were assembled. Relaxed molecular clock phylogenetic analysis demonstrated a molecular evolutionary rate for the Boende strain 4 to 10× slower than that of other Ebola lineages. These results demonstrate the utility of mNGS in broad-based pathogen detection and outbreak surveillance

Upconversion Nanomaterials: Synthesis, Mechanism, and Applications in Sensing

Upconversion is an optical process that involves the conversion of lower-energy photons into higher-energy photons. It has been extensively studied since mid-1960s and widely applied in optical devices. Over the past decade, high-quality rare earth-doped upconversion nanoparticles have been successfully synthesized with the rapid development of nanotechnology and are becoming more prominent in biological sciences. The synthesis methods are usually phase-based processes, such as thermal decomposition, hydrothermal reaction, and ionic liquids-based synthesis. The main difference between upconversion nanoparticles and other nanomaterials is that they can emit visible light under near infrared irradiation. The near infrared irradiation leads to low autofluorescence, less scattering and absorption, and deep penetration in biological samples. In this review, the synthesis of upconversion nanoparticles and the mechanisms of upconversion process will be discussed, followed by their applications in different areas, especially in the biological field for biosensing

Regulation of Myosin VI transport, tethering to actin and cargo binding

Myosin VI is a molecular motor that harnesses the conformational changes caused by ATP hydrolysis to generate motion along the actin filament (F-actin). As with all myosins, the motor domain of myosin VI contains an actin binding site and nucleotide binding site. The lever arm of myosin VI is followed by a coiled coil motif that mediates dimerization, and a cargo binding tail domain. Myosin VI is the only myosin that moves to the minus-end of F-actin. It has been implicated in multiple functions in disparate model systems. The ability of myosin VI to move processively across the actin filament provides a role for myosin VI in cargo transport, including uncoated endocytic vesicle (UCV) transport. Alternatively, kinetic studies have shown that myosin VI can be made to spend more of its actomyosin cycle tightly bound to actin when under increased load conditions, allowing myosin VI to also serve as a tension sensor that tethers membrane and protein elements to the actin cytoskeleton. The regulation of myosin VI at the level of the motor for it to act as a motor or tether is an intriguing possibility. In addition, myosin VI is mainly found in a cytoplasmic pool suggesting that its docking to cargoes is regulated, separately from its interaction with actin. In Chapter One, I show that myosin VI functions to transport uncoated endocytic vesicles (UCV) through the actin meshwork in our model retinal pigmented epithelial cell system, and in doing so demonstrate that the cortical actin meshwork acts as a barrier to uncoated endocytic vesicle trafficking to the pericentriolar region. In Chapter Two, I provide evidence that in vivo, myosin VI is regulated to act either as a transporter or a tension sensing anchor to actin following alteration at threonine 406. Finally in Chapter Three, I delineate a mechanism for myosin VI recruitment to the UCV cargo through its adapter GIPC/synectin. I posit that recruitment occurs via activation of the GIPC/synectin PDZ domain by PDZ motif- containing receptors being trafficked through the UC

Uncoated Endocytic Vesicles Require the Unconventional Myosin, Myo6, for Rapid Transport through Actin Barriers

After clathrin-mediated endocytosis, clathrin removal yields an uncoated vesicle population primed for fusion with the early endosome. Here we present the first characterization of uncoated vesicles and show that myo6, an unconventional myosin, functions to move these vesicles out of actin-rich regions found in epithelial cells. Time-lapse microscopy revealed that myo6-associated uncoated vesicles were motile and exhibited fusion and stretching events before endosome delivery, processes that were dependent on myo6 motor activity. In the absence of myo6 motor activity, uncoated vesicles remained trapped in the actin mesh, where they exhibited Brownian-like motion. Exit from the actin mesh occurred by a slow diffusion-based mechanism, delaying transferrin trafficking to the early endosome. Expression of a myo6 mutant that bound tightly to F-actin produced immobilized vesicles and blocked trafficking. Depolymerization of the actin cytoskeleton rescued this block and specifically accelerated transferrin delivery to the early endosome without affecting earlier steps in endocytosis. Therefore actin is a physical barrier impeding uncoated vesicle trafficking, and myo6 is recruited to move the vesicles through this barrier for fusion with the early endosome