Robust and rapid partitioning in thermoplastic.

Partitioning is the core technology supporting digital assays. It divides a sample into thousands of individual reactors prior to amplification and absolute quantification of target molecules. Thermoplastics are attractive materials for large scale manufacturing, however they have been seldomly used for fabricating partitioning arrays. Patitioning in thermoplastic devices has proven difficult due to the challenge of efficiently displacing the air trapped in the nanoliter structures during priming of thousands of chambers. Here, we report the design of an array of chambers made of thermoplastics where the progression of the liquid-air interface is controlled by capillary effects. Our device performs robust partitioning over a wide range of pressures and can be actuated at low pressure by a simple micropipette. Our thermoplastic device lays the foundation to cost-effective and instrument-free partitioning platforms, which could be deployed in low-resource settings.

A multicenter virome analysis of blood, feces, and saliva in myalgic encephalomyelitis/chronic fatigue syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is estimated to affect 0.4%-2.5% of the global population. Most cases are unexplained; however, some patients describe an antecedent viral infection or response to antiviral medications. We report here a multicenter study for the presence of viral nucleic acid in blood, feces, and saliva of patients with ME/CFS using polymerase chain reaction and high-throughput sequencing. We found no consistent group-specific differences other than a lower prevalence of anelloviruses in cases compared to healthy controls. Our findings suggest that future investigations into viral infections in ME/CFS should focus on adaptive immune responses rather than surveillance for viral gene products.

Nipah virus dynamics in bats and implications for spillover to humans.

Nipah virus (NiV) is an emerging bat-borne zoonotic virus that causes near-annual outbreaks of fatal encephalitis in South Asia-one of the most populous regions on Earth. In Bangladesh, infection occurs when people drink date-palm sap contaminated with bat excreta. Outbreaks are sporadic, and the influence of viral dynamics in bats on their temporal and spatial distribution is poorly understood. We analyzed data on host ecology, molecular epidemiology, serological dynamics, and viral genetics to characterize spatiotemporal patterns of NiV dynamics in its wildlife reservoir, Pteropus medius bats, in Bangladesh. We found that NiV transmission occurred throughout the country and throughout the year. Model results indicated that local transmission dynamics were modulated by density-dependent transmission, acquired immunity that is lost over time, and recrudescence. Increased transmission followed multiyear periods of declining seroprevalence due to bat-population turnover and individual loss of humoral immunity. Individual bats had smaller host ranges than other Pteropus species (spp.), although movement data and the discovery of a Malaysia-clade NiV strain in eastern Bangladesh suggest connectivity with bats east of Bangladesh. These data suggest that discrete multiannual local epizootics in bat populations contribute to the sporadic nature of NiV outbreaks in South Asia. At the same time, the broad spatial and temporal extent of NiV transmission, including the recent outbreak in Kerala, India, highlights the continued risk of spillover to humans wherever they may interact with pteropid bats and the importance of limiting opportunities for spillover throughout Pteropus's range.

Enzymatic-based cytometry, a sensitive single-cell cytometric method to assess BCR-ABL1 activity in CML.

We developed a simple, rapid and cost-effective enzymatic-based cytometry platform to measure intracellular signaling pathway activity. Our single-cell microwell array platform quantifies protein phosphorylation using enzymatic signal amplification and exploiting Michaelis-Menten kinetics. Our method provides a two-fold increase in resolution compared to conventional flow cytometry.

dPCR: A Technology Review.

Digital Polymerase Chain Reaction (dPCR) is a novel method for the absolute quantification of target nucleic acids. Quantification by dPCR hinges on the fact that the random distribution of molecules in many partitions follows a Poisson distribution. Each partition acts as an individual PCR microreactor and partitions containing amplified target sequences are detected by fluorescence. The proportion of PCR-positive partitions suffices to determine the concentration of the target sequence without a need for calibration. Advances in microfluidics enabled the current revolution of digital quantification by providing efficient partitioning methods. In this review, we compare the fundamental concepts behind the quantification of nucleic acids by dPCR and quantitative real-time PCR (qPCR). We detail the underlying statistics of dPCR and explain how it defines its precision and performance metrics. We review the different microfluidic digital PCR formats, present their underlying physical principles, and analyze the technological evolution of dPCR platforms. We present the novel multiplexing strategies enabled by dPCR and examine how isothermal amplification could be an alternative to PCR in digital assays. Finally, we determine whether the theoretical advantages of dPCR over qPCR hold true by perusing studies that directly compare assays implemented with both methods.

Click Chemistry Approaches to Expand the Repertoire of PEG-based Fluorinated Surfactants for Droplet Microfluidics.

We report the novel and simplified synthesis of fluorinated surfactants for droplet microfluidics. The range of applications of droplet microfluidics has greatly expanded during the last decade thanks to its ability to manipulate and process tiny amount of sample and reagents at high throughput in independent reactors. A critical component of the technology is the formulation of the immiscible oil phase that contains surfactants to stabilize droplets. The success of droplet microfluidics relies mostly on a single fluorinated formulation that uses a PFPE-PEG tri-block surfactant. The synthesis of this surfactant is laborious and requires skills in synthetic chemistry preventing the wider community to explore the synthesis of alternate surfactants. We sought to provide a simplified synthesis for novel PFPE-PEG surfactants based on click chemistry approaches such as copper-catalyzed azide-alkyne cycloaddition (CuAAC) and UV-activated thiol-yne reactions. Our strategy is based on converting a moisture sensitive intermediate typically used in the synthesis of the tri-block PFPE-PEG surfactant into a stable and click ready molecule. We successfully combined that fluorinated tail with differently functionalized PEG and glycerol ethoxylate molecules to generate surfactants with diverse structures via CuACC and thiol-yne reactions. We report the characterization, biocompatibility and ability to stabilize emulsions of those surfactants, as well as the unique advantages and challenges of the strategy.

Correction: Click chemistry approaches to expand the repertoire of PEG-based fluorinated surfactants for droplet microfluidics.

[This corrects the article DOI: 10.1039/C8RA01254G.].

Viral Encephalitis of Unknown Cause: Current Perspective and Recent Advances.

Viral encephalitis causes acute inflammation of the brain parenchyma and is a significant cause of human morbidity and mortality. Although Herpes Simplex encephalitis is the most frequent known cause of fatal sporadic encephalitis in humans, an increasingly wide range of viruses and other microbial pathogens are implicated. Up to 60% of cases of presumed viral encephalitis remain unexplained due to the failure of conventional laboratory techniques to detect an infectious agent. High-throughput DNA sequencing technologies have the potential to detect any microbial nucleic acid present in a biological specimen without any prior knowledge of the target sequence. While there remain challenges intrinsic to these technologies, they have great promise in virus discovery in unexplained encephalitis.

Pathosphere.org: pathogen detection and characterization through a web-based, open source informatics platform.

BACKGROUND: The detection of pathogens in complex sample backgrounds has been revolutionized by wide access to next-generation sequencing (NGS) platforms. However, analytical methods to support NGS platforms are not as uniformly available. Pathosphere (found at Pathosphere.org) is a cloud - based open - sourced community tool that allows for communication, collaboration and sharing of NGS analytical tools and data amongst scientists working in academia, industry and government. The architecture allows for users to upload data and run available bioinformatics pipelines without the need for onsite processing hardware or technical support. RESULTS: The pathogen detection capabilities hosted on Pathosphere were tested by analyzing pathogen-containing samples sequenced by NGS with both spiked human samples as well as human and zoonotic host backgrounds. Pathosphere analytical pipelines developed by Edgewood Chemical Biological Center (ECBC) identified spiked pathogens within a common sample analyzed by 454, Ion Torrent, and Illumina sequencing platforms. ECBC pipelines also correctly identified pathogens in human samples containing arenavirus in addition to animal samples containing flavivirus and coronavirus. These analytical methods were limited in the detection of sequences with limited homology to previous annotations within NCBI databases, such as parvovirus. Utilizing the pipeline-hosting adaptability of Pathosphere, the analytical suite was supplemented by analytical pipelines designed by the United States Army Medical Research Insititute of Infectious Diseases and Walter Reed Army Institute of Research (USAMRIID-WRAIR). These pipelines were implemented and detected parvovirus sequence in the sample that the ECBC iterative analysis previously failed to identify. CONCLUSIONS: By accurately detecting pathogens in a variety of samples, this work demonstrates the utility of Pathosphere and provides a platform for utilizing, modifying and creating pipelines for a variety of NGS technologies developed to detect pathogens in complex sample backgrounds. These results serve as an exhibition for the existing pipelines and web-based interface of Pathosphere as well as the plug-in adaptability that allows for integration of newer NGS analytical software as it becomes available.

Detection of zoonotic pathogens and characterization of novel viruses carried by commensal Rattus norvegicus in New York City.

Norway rats (Rattus norvegicus) are globally distributed and concentrate in urban environments, where they live and feed in closer proximity to human populations than most other mammals. Despite the potential role of rats as reservoirs of zoonotic diseases, the microbial diversity present in urban rat populations remains unexplored. In this study, we used targeted molecular assays to detect known bacterial, viral, and protozoan human pathogens and unbiased high-throughput sequencing to identify novel viruses related to agents of human disease in commensal Norway rats in New York City. We found that these rats are infected with bacterial pathogens known to cause acute or mild gastroenteritis in people, including atypical enteropathogenic Escherichia coli, Clostridium difficile, and Salmonella enterica, as well as infectious agents that have been associated with undifferentiated febrile illnesses, including Bartonella spp., Streptobacillus moniliformis, Leptospira interrogans, and Seoul hantavirus. We also identified a wide range of known and novel viruses from groups that contain important human pathogens, including sapoviruses, cardioviruses, kobuviruses, parechoviruses, rotaviruses, and hepaciviruses. The two novel hepaciviruses discovered in this study replicate in the liver of Norway rats and may have utility in establishing a small animal model of human hepatitis C virus infection. The results of this study demonstrate the diversity of microbes carried by commensal rodent species and highlight the need for improved pathogen surveillance and disease monitoring in urban environments. Importance: The observation that most emerging infectious diseases of humans originate in animal reservoirs has led to wide-scale microbial surveillance and discovery programs in wildlife, particularly in the developing world. Strikingly, less attention has been focused on commensal animals like rats, despite their abundance in urban centers and close proximity to human populations. To begin to explore the zoonotic disease risk posed by urban rat populations, we trapped and surveyed Norway rats collected in New York City over a 1-year period. This analysis revealed a striking diversity of known pathogens and novel viruses in our study population, including multiple agents associated with acute gastroenteritis or febrile illnesses in people. Our findings indicate that urban rats are reservoirs for a vast diversity of microbes that may affect human health and indicate a need for increased surveillance and awareness of the disease risks associated with urban rodent infestation.

Use of staged molecular analysis to determine causes of unexplained central nervous system infections.

No agent is implicated in most central nervous system (CNS) infections. To investigate cerebrospinal fluid samples from patients with CNS infections of unknown cause in 1 hospital in Taiwan, we used a staged molecular approach, incorporating techniques including multiplex MassTag PCR, 16S rRNA PCR, DNA microarray, and high-throughput pyrosequencing. We determined the infectious agent for 31 (24%) of 131 previously negative samples. Candidate pathogens were identified for 25 (27%) of 94 unexplained meningitis cases and 6 (16%) of 37 unexplained encephalitis cases. Epstein-Barr virus (18 infections) accounted for most of the identified agents in unexplained meningitis cases, followed by Escherichia coli (5), enterovirus (2), human herpesvirus 2 (1), and Mycobacterium tuberculosis. Herpesviruses were identified in samples from patients with unexplained encephalitis cases, including varicella-zoster virus (3 infections), human herpesvirus 1 (2), and cytomegalovirus (1). Our study confirms the power of multiplex MassTag PCR as a rapid diagnostic tool for identifying pathogens causing unexplained CNS infections.

Bats are a major natural reservoir for hepaciviruses and pegiviruses.

Although there are over 1,150 bat species worldwide, the diversity of viruses harbored by bats has only recently come into focus as a result of expanded wildlife surveillance. Such surveys are of importance in determining the potential for novel viruses to emerge in humans, and for optimal management of bats and their habitats. To enhance our knowledge of the viral diversity present in bats, we initially surveyed 415 sera from African and Central American bats. Unbiased high-throughput sequencing revealed the presence of a highly diverse group of bat-derived viruses related to hepaciviruses and pegiviruses within the family Flaviridae. Subsequent PCR screening of 1,258 bat specimens collected worldwide indicated the presence of these viruses also in North America and Asia. A total of 83 bat-derived viruses were identified, representing an infection rate of nearly 5%. Evolutionary analyses revealed that all known hepaciviruses and pegiviruses, including those previously documented in humans and other primates, fall within the phylogenetic diversity of the bat-derived viruses described here. The prevalence, unprecedented viral biodiversity, phylogenetic divergence, and worldwide distribution of the bat-derived viruses suggest that bats are a major and ancient natural reservoir for both hepaciviruses and pegiviruses and provide insights into the evolutionary history of hepatitis C virus and the human GB viruses.

Genetic characterization of K13965, a strain of Oak Vale virus from Western Australia.

K13965, an uncharacterized virus, was isolated in 1993 from Anopheles annulipes mosquitoes collected in the Kimberley region of northern Western Australia. Here, we report its genomic sequence, identify it as a rhabdovirus, and characterize its phylogenetic relationships. The genome comprises a P' (C) and SH protein similar to the recently characterized Tupaia and Durham viruses, and shows overlap between G and L genes. Comparison of K13965 genome sequence to other rhabdoviruses identified K13965 as a strain of the unclassified Australian Oak Vale rhabdovirus, whose complete genome sequence we also determined. Phylogenetic analysis of N and L sequences indicated genetic relationship to a recently proposed Sandjima virus clade, although the Oak Vale virus sequences form a branch separate from the African members of that group.

An insect nidovirus emerging from a primary tropical rainforest.

Tropical rainforests show the highest level of terrestrial biodiversity and may be an important contributor to microbial diversity. Exploitation of these ecosystems may foster the emergence of novel pathogens. We report the discovery of the first insect-associated nidovirus, tentatively named Cavally virus (CAVV). CAVV was found with a prevalence of 9.3% during a survey of mosquito-associated viruses along an anthropogenic disturbance gradient in Côte d'Ivoire. Analysis of habitat-specific virus diversity and ancestral state reconstruction demonstrated an origin of CAVV in a pristine rainforest with subsequent spread into agriculture and human settlements. Virus extension from the forest was associated with a decrease in virus diversity (P<0.01) and an increase in virus prevalence (P<0.00001). CAVV is an enveloped virus with large surface projections. The RNA genome comprises 20,108 nucleotides with seven major open reading frames (ORFs). ORF1a and -1b encode two large proteins that share essential features with phylogenetically higher representatives of the order Nidovirales, including the families Coronavirinae and Torovirinae, but also with families in a basal phylogenetic relationship, including the families Roniviridae and Arteriviridae. Genetic markers uniquely conserved in nidoviruses, such as an endoribonuclease- and helicase-associated zinc-binding domain, are conserved in CAVV. ORF2a and -2b are predicted to code for structural proteins S and N, respectively, while ORF3a and -3b encode proteins with membrane-spanning regions. CAVV produces three subgenomic mRNAs with 5' leader sequences (of different lengths) derived from the 5' end of the genome. This novel cluster of mosquito-associated nidoviruses is likely to represent a novel family within the order Nidovirales.

Identification of a severe acute respiratory syndrome coronavirus-like virus in a leaf-nosed bat in Nigeria.

Bats are reservoirs for emerging zoonotic viruses that can have a profound impact on human and animal health, including lyssaviruses, filoviruses, paramyxoviruses, and severe acute respiratory syndrome coronaviruses (SARS-CoVs). In the course of a project focused on pathogen discovery in contexts where human-bat contact might facilitate more efficient interspecies transmission of viruses, we surveyed gastrointestinal tissue obtained from bats collected in caves in Nigeria that are frequented by humans. Coronavirus consensus PCR and unbiased high-throughput pyrosequencing revealed the presence of coronavirus sequences related to those of SARS-CoV in a Commerson's leaf-nosed bat (Hipposideros commersoni). Additional genomic sequencing indicated that this virus, unlike subgroup 2b CoVs, which includes SARS-CoV, is unique, comprising three overlapping open reading frames between the M and N genes and two conserved stem-loop II motifs. Phylogenetic analyses in conjunction with these features suggest that this virus represents a new subgroup within group 2 CoVs.

Identification and characterization of a new bocavirus species in gorillas.

A novel parvovirus, provisionally named Gorilla Bocavirus species 1 (GBoV1), was identified in four stool samples from Western gorillas (Gorilla gorilla) with acute enteritis. The complete genomic sequence of the new parvovirus revealed three open reading frames (ORFs) with an organization similar to that of known bocaviruses. Phylogenetic analysis using complete capsid and non structural (NS) gene sequence suggested that the new parvovirus is most closely related to human bocaviruses (HBoV). However, the NS ORF is more similar in length to the NS ORF found in canine minute virus and bovine parvovirus than in HBoV. Comparative genetic analysis using GBoV and HBoV genomes enabled characterization of unique splice donor and acceptor sites that appear to be highly conserved among all four HBoV species, and provided evidence for expression of two different NS proteins in all primate bocaviruses. GBoV is the first non-human primate bocavirus identified and provides new insights into the genetic diversity and evolution of this highly prevalent and recently discovered group of parvoviruses.

Identification of GBV-D, a novel GB-like flavivirus from old world frugivorous bats (Pteropus giganteus) in Bangladesh.

Bats are reservoirs for a wide range of zoonotic agents including lyssa-, henipah-, SARS-like corona-, Marburg-, Ebola-, and astroviruses. In an effort to survey for the presence of other infectious agents, known and unknown, we screened sera from 16 Pteropus giganteus bats from Faridpur, Bangladesh, using high-throughput pyrosequencing. Sequence analyses indicated the presence of a previously undescribed virus that has approximately 50% identity at the amino acid level to GB virus A and C (GBV-A and -C). Viral nucleic acid was present in 5 of 98 sera (5%) from a single colony of free-ranging bats. Infection was not associated with evidence of hepatitis or hepatic dysfunction. Phylogenetic analysis indicates that this first GBV-like flavivirus reported in bats constitutes a distinct species within the Flaviviridae family and is ancestral to the GBV-A and -C virus clades.

Nonparametric methods for the analysis of single-color pathogen microarrays.

BACKGROUND: The analysis of oligonucleotide microarray data in pathogen surveillance and discovery is a challenging task. Target template concentration, nucleic acid integrity, and host nucleic acid composition can each have a profound effect on signal distribution. Exploratory analysis of fluorescent signal distribution in clinical samples has revealed deviations from normality, suggesting that distribution-free approaches should be applied. RESULTS: Positive predictive value and false positive rates were examined to assess the utility of three well-established nonparametric methods for the analysis of viral array hybridization data: (1) Mann-Whitney U, (2) the Spearman correlation coefficient and (3) the chi-square test. Of the three tests, the chi-square proved most useful. CONCLUSIONS: The acceptance of microarray use for routine clinical diagnostics will require that the technology be accompanied by simple yet reliable analytic methods. We report that our implementation of the chi-square test yielded a combination of low false positive rates and a high degree of predictive accuracy.

Astrovirus encephalitis in boy with X-linked agammaglobulinemia.

Encephalitis is a major cause of death worldwide. Although >100 pathogens have been identified as causative agents, the pathogen is not determined for up to 75% of cases. This diagnostic failure impedes effective treatment and underscores the need for better tools and new approaches for detecting novel pathogens or determining new manifestations of known pathogens. Although astroviruses are commonly associated with gastroenteritis, they have not been associated with central nervous system disease. Using unbiased pyrosequencing, we detected an astrovirus as the causative agent for encephalitis in a 15-year-old boy with agammaglobulinemia; several laboratories had failed to identify the agent. Our findings expand the spectrum of causative agents associated with encephalitis and highlight unbiased molecular technology as a valuable tool for differential diagnosis of unexplained disease.