Salivary Detection of Dengue Virus NS1 Protein with a Label-Free Immunosensor for Early Dengue Diagnosis.

Dengue virus (DENV) is a highly pathogenic, arthropod-borne virus transmitted between people by Aedes mosquitoes. Despite efforts to prevent global spread, the potential for DENV epidemics is increasing world-wide. Annually, 3.6 billion people are at risk of infection. With no licensed vaccine, early diagnosis of dengue infection is critical for clinical management and patient survival. Detection of DENV non-structural protein 1 (NS1) is a clinically accepted biomarker for the early detection of DENV infection. Unfortunately, virtually all of the laboratory and commercial DENV NS1 diagnostic methods require a blood draw for sample analysis, limiting point-of-care diagnostics and decreases patient willingness. Alternatively, NS1 in human saliva has been identified for the potential early diagnosis of DENV infection. The collection of saliva is simple, non-invasive, painless, and inexpensive, even by minimally trained personnel. In this study, we present a label-free chemiresistive immunosensor for the detection of the DENV NS1 protein utilizing a network of single-walled carbon nanotubes functionalized with anti-dengue NS1 monoclonal antibodies. NS1 was successfully detected in adulterated artificial human saliva over the range of clinically relevant concentrations with high sensitivity and selectivity. It has potential application in clinical diagnosis and the ease of collection allows for self-testing, even within the home.

Point-of-Use Nanobiosensor for Detection of Dengue Virus NS1 Antigen in Adult Aedes aegypti: A Potential Tool for Improved Dengue Surveillance.

Dengue virus (DENV) and its primary mosquito vectors Aedes spp. have spread to every continent except Antarctica, causing outbreaks and autochthonous transmission in previously disease-free regions. Recently, the spread of other arboviruses carried by invasive Aedes spp., such as Chikungunya and Zika, seem to be following similar trends as DENV and have renewed interest in monitoring and modeling arboviruses within mosquito vectors. Unfortunately, current commercially available detection methods are designed for the diagnosis of DENV in humans or are too expensive and complex for sustainable monitoring. We report a novel electronic nanobiosensor utilizing a single-walled carbon nanotube networks chemiresistor transducer functionalized with antidengue NS1 monoclonal antibodies for rapid detection of the dengue nonstructural protein 1 (NS1). NS1 is a highly conserved protein secreted at high concentrations during viral replication and is a biomarker for DENV infection. NS1 was successfully detected in spiked adult Aedes aegypti homogenate over a broad dynamic range with high sensitivity and selectivity. The biosensor is compatible with "gold-standard" adult mosquito field-collection protocols and generates electronic data that can be readily stored or wirelessly transmitted. Thus, it has potential for remote and real-time monitoring of wild mosquito populations.

A heparin-functionalized carbon nanotube-based affinity biosensor for dengue virus.

Dengue virus is an arthropod-borne virus transmitted primarily by Aedes mosquitos and is major cause of disease in tropical and subtropical regions. Colloquially known as Dengue Fever, infection can cause hemorrhagic disorders and death in humans and non-human primates. We report a novel electronic biosensor based on a single-walled carbon nanotube network chemiresistive transducer that is functionalized with heparin for low-cost, label-free, ultra-sensitive, and rapid detection of whole dengue virus (DENV). Heparin, an analog of the heparan sulfate proteoglycans that are receptors for dengue virus during infection of Vero cells and hepatocytes, was used for the first time in a biosensor as a biorecognition element instead of traditional antibody. Detection of DENV in viral culture supernatant has similar sensitivity as the corresponding viral titer in phosphate buffer despite the presence of growth media and Vero cell lysate. The biosensor demonstrated sensitivity within the clinically relevant range for humans and infected Aedes aegypti. It has potential application in clinical diagnosis and can improve point-of-care diagnostics of dengue infection.

Determining the Solar Inactivation Rate of BK Polyomavirus by Molecular Beacon.

The application of molecular beacons (MB) that bind to precise sequences of mRNA provides a near-universal approach in detecting evidence of viral replication. Here, we demonstrate the detection of BK Polyomavirus (BKPyV), an emerging indicator of microbiological water quality, by a quantum dot-based MB. The MB allowed us to rapidly characterize the inactivation rate of BKPyV following exposure to a solar simulator (kobs = 0.578 ± 0.024 h(-1), R(2) = 0.92). Results were validated through a traditional cell-culture assay with immunofluorescence detection (kobs = 0.568 ± 0.011 h(-1), R(2) = 0.97), which exhibited a strong correlation to MB data (R(2) = 0.93). Obtaining solar inactivation rates for BKPyV demonstrates the first use of a MB in characterizing a microbiological inactivation profile and helps assess the appropriateness of adopting BKPyV as an indicator organism for water quality.

Long-term characterization of residential runoff and assessing potential surrogates of fecal indicator organisms.

Investigations into the microbiological impacts of urban runoff on receiving water bodies, especially during storm conditions, have yielded general paradigms that influence runoff abatement and control management strategies. To determine whether these trends are present in other runoff sources, the physical, chemical, and microbiological components of residential runoff from eight neighborhoods in Northern and Southern California were characterized over the course of five years. Sampling occurred regularly and during storm events, resulting in 833 data sets. Analysis of runoff data assisted in characterizing residential runoff, elucidating differences between dry and storm conditions, and identifying surrogates capable of assessing microbiological quality. Results indicate that although microbial loading increases during storm events similar to urban runoff, annual microbial loading in these study sites principally occurs during dry conditions (24% storm, 76% dry). Generated artificial neural network and multiple linear regression models assessed surrogate performance by accurately predicting Escherichia coli concentrations from validation data sets (R(2) = 0.74 and 0.77, respectively), but required input from other fecal indicator organism (FIO) variables to maintain performance (R(2) = 0.27 and 0.18, respectively, without FIO). This long-term analysis of residential runoff highlights characteristics distinct from urban runoff and establishes necessary variables for determining microbiological quality, thus better informing future management strategies.

Cryptosporidium infection, onsite wastewater systems and private wells in the arid Southwest.

Few prior studies have examined the potential health risks from transmission of enteric parasites via aquifers contaminated by wastewater from onsite systems. A cross-sectional study of 600 residents in households served with either onsite wastewater systems and private wells or city sewer/water systems in three different sites in central New Mexico compared serological responses to Cryptosporidium, a common waterborne infections agent. Study participants completed a short self-administered questionnaire with questions on demographic characteristics, characteristics of the onsite wastewater system and private well, and common risk factors associated with cryptosporidiosis. A sample of household tap water was collected, as well as a blood sample from each study participant to measure IgG responses to antigen groups for Cryptosporidium. Logistic regression analysis showed a significant association between having an onsite wastewater system and private well and the 27-kDa marker for Cryptosporidium in the River Valley site after adjusting for covariates (OR = 1.98; 95% CI = 1.11-3.55). This study, together with one prior study, suggests that the presence of onsite wastewater systems and private wells might be associated with an increased risk of Cryptosporidium infection.

Quantitative assessment of in vivo HIV protease activity using genetically engineered QD-based FRET probes.

HIV protease plays a central role in its life cycle leading to release of functional viral particles. It has been successfully used as a therapeutic target to block HIV infection. Several protease inhibitors (PIs) are currently being employed as a part of anti-HIV therapy. However, the constant genetic drift in the virus leads to accumulation of mutations in both cleavage site and the protease, resulting in resistance and failure of therapy. We reported the use of a quantum dot (QD)-based protein probe for the in vivo monitoring of HIV-1 protease activity based on fluorescence resonance energy transfer. In the current study, we demonstrate the utility of this approach by quantifying the in vivo cleavage rates of three known protease and cleavage site mutations in the presence or absence of different PIs. The changes in IC50 values for the different PIs were similar to that observed in patients, validating our assay as a rapid platform for PI screening.

Use of flow cytometry for rapid, quantitative detection of poliovirus-infected cells via TAT peptide-delivered molecular beacons.

Rapid and efficient detection of viral infection is crucial for the prevention of disease spread during an outbreak and for timely clinical management. In this paper, the utility of Tat peptide-modified molecular beacons (MBs) as a rapid diagnostic tool for the detection of virus-infected cells was demonstrated. The rapid intracellular delivery mediated by the Tat peptide enabled the detection of infected cells within 30 s, reaching saturation in signal in 30 min. This rapid detection scheme was coupled with flow cytometry (FC), resulting in an automated, high-throughput method for the identification of virus-infected cells. Because of the 2-order-of-magnitude difference in fluorescence intensity between infected and uninfected cells, as few as 1% infected cells could be detected. Because of its speed and sensitivity, this approach may be adapted for the practical diagnosis of multiple viral infections.

Simultaneous detection of infectious human echoviruses and adenoviruses by an in situ nuclease-resistant molecular beacon-based assay.

A multiplex methodology using two nuclease-resistant molecular beacons that target specific genomic regions of adenovirus 2 and echovirus 17 during simultaneous infection in A549 cells is presented. Using fluorescence microscopy, visualization of enteroviral and adenoviral replication was possible within 3 h postinfection.

Effects of residual antibiotics in groundwater on Salmonella typhimurium: changes in antibiotic resistance, in vivo and in vitro pathogenicity.

An outbreak-causing strain of Salmonella enterica serovar Typhimurium was exposed to groundwater with residual antibiotics for up to four weeks. Representative concentrations (0.05, 1, and 100 µg L(-1)) of amoxicillin, tetracycline, and a mixture of several other antibiotics (1 µg L(-1) each) were spiked into artificially prepared groundwater (AGW). Antibiotic susceptibility analysis and the virulence response of stressed Salmonella were determined on a weekly basis by using human epithelial cells (HEp2) and soil nematodes (C. elegans). Results have shown that Salmonella typhimurium remains viable for long periods of exposure to antibiotic-supplemented groundwater; however, they failed to cultivate as an indication of a viable but nonculturable state. Prolonged antibiotics exposure did not induce any changes in the antibiotic susceptibility profile of the S. typhimurium strain used in this study. S. typhimurium exposed to 0.05 and 1 µg L(-1) amoxicillin, and 1 µg L(-1) tetracycline showed hyper-virulent profiles in both in vitro and in vivo virulence assays with the HEp2 cells and C. elegans respectively, most evident following 2nd and 3rd weeks of exposure.

Detection of murine norovirus-1 by using TAT peptide-delivered molecular beacons.

A TAT peptide-delivered molecular beacon was developed and utilized to enumerate murine norovirus 1, a human norovirus (NoV) surrogate, in RAW 264.7 cells. This allowed the detection of a single infective virus within 6 h, a 12-fold improvement in time required for viral detection and quantification compared to that required by the conventional plaque assay.

Detecting RNA viruses in living mammalian cells by fluorescence microscopy.

Traditional methods that rely on viral isolation and culture techniques continue to be the gold standards used for detection of infectious viral particles. However, new techniques that rely on visualization of live cells can shed light on understanding virus-host interaction for early stage detection and potential drug discovery. Live-cell imaging techniques that incorporate fluorescent probes into viral components provide opportunities for understanding mRNA expression, interaction, and virus movement and localization. Other viral replication events inside a host cell can be exploited for non-invasive detection, such as single-virus tracking, which does not inhibit viral infectivity or cellular function. This review highlights some of the recent advances made using these novel approaches for visualization of viral entry and replication in live cells.

A quantum-dot based protein module for in vivo monitoring of protease activity through fluorescence resonance energy transfer.

Here, we present a new generation of nanoscale probes for in vivo monitoring of protease activity by fluorescence resonance energy transfer (FRET). The approach is based on a genetically programmable protein module carrying a fluorescently labeled, protease-specific sequence that can self-assemble onto quantum dots. The protein module was used for real-time detection of human immunodeficiency virus type-1 protease (HIV-1 Pr) activity as well as quantitative assessment of inhibitor efficiency.

A fluorescence resonance energy transfer-based fluorometer assay for screening anti-coxsackievirus B3 compounds.

In view of the need to develop a simple and rapid method to screen for antiviral therapeutic agents, a fluorescence resonance energy transfer (FRET)-based reporter system consisting of engineered mammalian cells expressing a cyan fluorescent protein-yellow fluorescent protein (CFP-YFP) pair linked by a short peptide containing the cleavage site of viral protease 2A (2A(pro)) was developed. By detecting the 2A(pro) produced early during the virus infection cycle, the CFP-YFP pair effectively identifies infectious coxsackievirus B3 (CVB3), a picornavirus that causes viral myocarditis in humans. The reporter system was used to screen a library of 2000 drugs and natural products for potential antiviral compounds. The reporter cells were treated with the test compounds, challenged with CVB3, and then examined using a fluorometer at 24h post-infection. Sixty-four compounds, mostly therapeutic drugs, antimicrobial compounds and compounds with unknown functions, caused at least 50% inhibition of 2A(pro) activity. Three known antiviral compounds, cosmosiin, ribavirin and baicalein, were also identified in the screening. The developed method is an effective strategy for rapid screening, and identifies compounds that inhibit CVB3 2A(pro). This method should be a valuable aid in the antiviral drug discovery effort.

Label-free chemiresistive immunosensors for viruses.

We report development, characterization, and testing of chemiresistive immunosensors based on single polypyrrole (Ppy) nanowire for highly sensitive, specific, label free, and direct detection of viruses. Bacteriophages T7 and MS2 were used as safe models for viruses for demonstration. Ppy nanowires were electrochemically polymerized into alumina template, and single nanowire based devices were assembled on a pair of gold electrodes by ac dielectrophoretic alignment and anchored using maskless electrodeposition. Anti-T7 or anti-MS2 antibodies were immobilized on single Ppy nanowire using EDC-NHS chemistry to fabricate nanobiosensor for the detection of corresponding bacteriophage. The biosensors showed excellent sensitivity with a lower detection limit of 10(-3) plaque forming unit (PFU) in 10 mM phosphate buffer, wide dynamic range and excellent selectivity. The immunosensors were successfully applied for the detection of phages in spiked untreated urban runoff water samples. The results show the potential of these sensors in health care, environmental monitoring, food safety and homeland security for sensitive, specific, rapid, and affordable detection of bioagents/pathogens.

Kinetics of inactivation of indicator pathogens during thermophilic anaerobic digestion.

Thermophilic anaerobic sludge digestion is a promising process to divert waste to beneficial use, but an important question is the required temperature and holding time to achieve a given degree of pathogen inactivation. In this study, the kinetics of inactivation of Ascaris suum and vaccine strain poliovirus type 1 (PVS-1), selected as indicators for helminth ova and enteric viruses respectively, were determined during anaerobic digestion at temperatures ranging from 51 to 56 °C. Inactivation of both indicator organisms was fast with greater than two log reductions achieved within 2 h for A. suum and three log reductions for PVS-1, suggesting that the current U.S. regulations are largely conservative. The first-order inactivation rate constants k followed Arrhenius relationship with activation energies of 105 and 39 KJ mol(-1) for A. suum and PVS-1, respectively indicating that A. suum was more sensitive to temperature. Although inactivation was fast, the presence of compounds in the sludge that are known to be protective of pathogen inactivation was observed, suggesting that composition-dependent time-temperature relationships are necessary.

Carbon nanotubes-based chemiresistive biosensors for detection of microorganisms.

The paper reports carbon nanotube (CNT)-based immunosensors for the detection of two types of microorganisms, bacteria and viruses. The pathogen Escherichia coli O157:H7 and the bacteriophage T7 were selected as model for bacteria and viruses, respectively, while E. coli K12 and the bacteriophage MS2 were used to assess the selectivity of the biosensor. The transduction element consisted of single-walled carbon nanotubes aligned in parallel bridging two gold electrodes to function as a chemiresistive biosensor. Single-walled carbon nanotubes (SWNTs) were functionalized with specific antibodies (Ab) for the different microorganisms by covalent immobilization to the non-covalently bound 1-pyrene butanoic acid succinimidyl ester. A significant increase in the resistance of the device was observed when the biosensor was exposed to E. coli O157:H7 whole cells or lysates with a limit of detection of 10(5) and 10(3) CFU (colony forming units)/mL, corresponding to 10(3) and 10(1) CFU/chip, respectively, while no response was observed when the biosensor was exposed to the E. coli K12. In the case of virus detection, a significant resistance increase was detected due to interaction of the bacteriophages with the Abs, with a limit of detection of 10(3) PFU/mL corresponding to 10(1)PFU/chip and excellent selectivity against MS2 bacteriophage. The sensor exhibited a fast response time of ∼5 min in the case of bacteriophage detection, while the response time for the detection of bacteria was 60 min.

Molecular beacon-quantum dot-Au nanoparticle hybrid nanoprobes for visualizing virus replication in living cells.

Here we describe a new hybrid fluorescent nanoprobe composed of a nuclease-resistant molecular beacon (MB) backbone, CdSe-ZnS core-shell quantum dots (QDs) as donors, and gold nanoparticles (Au NPs) as quenchers, for the real-time visualization of virus replication in living cells. By using a Au NP-MB to QD ratio of 6 : 1, a 7.3-fold increase in fluorescent signal was achieved upon target binding. For living cell experiments, a hexahistidine-appended Tat peptide was self-assembled onto the QD surface to provide nearly 100% non-invasive delivery of the QD-MB-Au NP probes within 2 h. By directly visualizing the fluorescent complexes formed with the newly synthesized viral RNA, this QD-MB-Au NP probe provided sensitive and real-time detection of infectious viruses as well as the real-time visualization of cell-to-cell virus spreading.

Detection of infective poliovirus by a simple, rapid, and sensitive flow cytometry method based on fluorescence resonance energy transfer technology.

The rapid and effective detection of virus infection is critical for clinical management and prevention of disease spread during an outbreak. Several methods have been developed for this purpose, of which classical serological and viral nucleic acid detection are the most common. We describe an alternative approach that utilizes engineered cells expressing fluorescent proteins undergoing fluorescence resonance energy transfer (FRET) upon cleavage by the viral 2A protease (2A(pro)) as an indication of infection. Quantification of the infectious-virus titers was resolved by using flow cytometry, and utility was demonstrated for the detection of poliovirus 1 (PV1) infection. Engineered buffalo green monkey kidney (BGMK) cells expressing the cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) substrate linked by a cleavage recognition site for PV1 2A(pro) were infected with different titers of PV1. After incubation at various time points, cells were harvested, washed, and subjected to flow cytometry analysis. The number of infected cells was determined by counting the number of cells with an increased CFP-to-YFP ratio. As early as 5 h postinfection, a significant number of infected cells (3%) was detected by flow cytometry, and cells infected with only 1 PFU were detected after 12 h postinfection. When applied to an environmental water sample spiked with PV1, the flow cytometry-based assay provided a level of sensitivity similar to that of the plaque assay for detecting and quantifying infectious virus particles. This approach, therefore, is more rapid than plaque assays and can be used to detect other viruses that frequently do not form clear plaques on cell cultures.

Real-time molecular methods to detect infectious viruses.

Waterborne transmitted viruses pose a public health threat due to their stability in aquatic environment and the easy transmission with high morbidity rates at low infectious doses. Two major challenge of virus analysis include a lack of adequate information in infectivity and the inability to cultivate certain epidemiologically important viruses in vitro. The use of fluorescent probes in conjunction with fluorescence microscopy allows us to reveal dynamic interactions of the viruses with different cellular structures in living cells that are impossible to detect by immunological or PCR-based experiments. Real-time viral detection in vivo provides sufficient information regarding multiple steps in infection process at molecular level, which will be valuable for the prevention and control of viral infection.