The overall aim of this project was to develop a sensitive, specific and reliable molecular assay for the detection of human pathogenic viruses in shellfish. In initial studies, 'viral surrogates' were used to evaluate two different assay formats, the reverse transcriptase-polymerase chain reaction-enzyme-linked immunosorbent assay (RT-PCR-ELISA) and the enzyme-labelled deoxynucleic acid-enzyme-linked coagulation assay (EDNA-ELCA). Each format was tested for ease of use, reliability and sensitivity, when compared with ethidium bromide gel detection. The RT-PCR-ELISA proved to be a successful alternative to ethidium bromide gel electrophoresis and studies involving virus detection in contaminated environmental samples were performed. The newly developed ELISA method was able to successfully detect enterovirus (EV) and Norwalk-like viruses (NLVs) in artificially, and naturally contaminated shellfish. In shellfish studies the ELISA had a detection sensitivity of 10-100, and 100-1000 TCID50 PV respectively, when a traditional elution/precipitation, and an immunocapture procedure were employed. The assay could also successfully detect virus in trout kidney samples, artificially contaminated with infectious pancreatic necrosis virus (IPNV), with detection sensitivities of 105, and 107 pfu reported when the elution/precipitation, and immunocapture procedures were used. In naturally contaminated shellfish samples, positive NLV and EV detection were achieved using the ELISA method. The ability of the ELISA to positively detect virus in environmental samples was compared to the TaqMan quantitative PCR system, an alternative detection method. Both methods were used to screen various contaminated environmental samples, including faeces and shellfish. The ELISA performed well, and unlike the TaqMan system no optimisation for each sample matrix tested was required. Overall the ELISA was shown to be a very robust and sensitive method. The technique was easily established in a new laboratory and no specialised equipment was required to perform the assay. The method has a high sample throughput, capable of screening 96 samples per run. Each sample takes only approximately 50 s to screen, making the technique extremely time efficient. The ELISA is a safe, quick, reliable technique, which has great potential for use as a standard virus detection method in a standard equipped laboratory.

With the great promise that metagenomics holds in exploring virome composition and discovering novel virus species, there is a pressing demand for comprehensive and up-to-date reference databases to enhance the downstream bioinformatics analysis. In this study, a RNA virus reference database (RVRD) was developed by manual and computational curation of RNA virus genomes downloaded from the three major virus sequence databases including NCBI, ViralZone, and ViPR. To reduce viral sequence redundancy caused by multiple identical or nearly identical sequences, sequences were first clustered and all sequences except one in a cluster that have more than 98% identity to one another were removed. Other identity cutoffs were also examined, and Hepatitis C virus genomes were studied in detail as an example. Using the 98% identity cutoff, sequences obtained from ViPR were combined with the unique RNA virus references from NCBI and ViralZone to generate the final RVRD. The resulting RVRD contained 23,085 sequences, nearly 5 times the size of NCBI RNA virus reference, and had a broad coverage of RNA virus families, with significant expansion on circular ssRNA virus and pathogenic virus families. Compared to NCBI RNA virus reference in performance evaluation, using RVRD as reference database identified more RNA virus species in RNAseq data derived from wastewater samples. Moreover, using RVRD as reference database also led to the discovery of porcine rotavirus as the etiology of unexplained diarrhea observed in pigs. RVRD is publicly available for enhancing RNA virus metagenomics. / Master of Science / Next-generation sequencing technology has demonstrated capability for the detection of viruses in various samples, but one challenge in bioinformatics analysis is the lack of well-curated reference databases, especially for RNA viruses. In this study, a RNA virus reference database (RVRD) was developed by manual and computational curation from the three commonly used resources: NCBI, ViralZone, and ViPR. While RVRD was managed to be comprehensive with broad coverage of RNA virus families, clustering was performed to reduce redundant sequences. The performance of RVRD was compared with NCBI RNA virus reference database using the pipeline FastViromeExplorer developed by our lab recently, the results showed that more RNA viruses were identified in several metagenomic datasets using RVRD, indicating improved performance in practice.

Numerous toxins and pathogens gain entry into mammalian cells using cell surface glycans. The Iyer group at Georgia State University is working on the development of glycoconjugates for the accurate detection of infectious agents. In this thesis, I have focused on the development of glycans to detect influenza virus and norovirus. In the first section, I have focused on influenza viruses. A panel of synthetic glycans was synthesized as receptor mimics for the specific capture of influenza viruses. The synthetic glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. This glycan printed microarray was evaluated for its ability to capture three strains of influenza viruses. The analytical limit of detection is ~10 pfu/ml, (plaque forming units/milliliter) which is clinical relevant as 102 viral particles are typically required to cause infection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using the microarray and determined the feasibility of this system to determine antiviral resistance for different strains. In addition to optical detection, I developed an electrochemical assay to rapidly detect influenza viruses. Here, we utilized an unique property of influenza viral surface enzyme, Neuraminidase (NA), which cleaves terminal N-Acetyl Neuraminic acid (sialic acid) from cell surfaces and proteins. We designed an electrochemical assay that uses glucose bearing sialic acid substrates. Glucose is released when exposed to viral NA or intact viruses. The released glucose can be detected using repurposed glucose meters. Thus, personal glucose meters that were designed to assist diabetics and prediabetics monitor blood glucose can potentially be used to detect pathogens. Using this approach, we have detected 19 unique strains of influenza viruses. We also demonstrated drug susceptibility using this assay. The limit of detection of this assay is 102 pfu/sample, which is clinically relevant. The results were validated plaque assays and polymerase chain reaction (PCR). In the second part of this thesis, I focused on norovirus detection. I developed a focused glycan microarray that comprised of a library of histo blood group antigens (HBGAs). The HBGAs were attached to a carrier protein and printed onto activated glass slides. A panel of norovirus virus like particles (VLPs) and strains that included different genogroups was exposed to the microarray. We found that different VLPs and strains give rise to unique binding patterns. When the binding pattern of VLPs for a particular strain were compared to the corresponding intact virus, the binding patterns didn't match well, presumably because the virus does not recognize the same antibody as the VLPs. Unfortunately, antibodies for the virus cannot be generated because the virus cannot be grown in a laboratory setting. Indeed, all norovirus samples are obtained from human challenge studies. I also used surface plasmon resonance (SPR) studies in an effort to determine the binding affinities. Divalent biotinylated H type glycans were synthesized and their binding affinities with different VLPs and viral strains were determined. Initial studies suggest that the binding affinities are strain specific. These results demonstrate that glycans can be used to capture and isolate norovirus, although more research is required to develop glycan based norovirus detection kits.

This thesis reports on the development of a novel mass spectrometer combining image charge detection with surface induced dissociation for real-time analysis of intact viruses. Protonated viruses produced using electrospray are accelerated and subsequently impact on a solid surface. Capsid peptides released during the impact are analyzed using time-of-flight mass spectrometry. Image charge detection is used to measure the mass and charge states of structurally intact, electrosprayed viruses prior to impact. Since virus capsids are composed of loosely-bound proteins, collision of viruses with surfaces at moderate impact energies could release intact proteins. The masses and numbers of different protein types combined with the mass of the intact virus represent a unique signature useful for accurate, real-time virus identification. The progress of instrumentation developed thus far is reported. Techniques were developed for electrospraying intact viruses, including electrospray capillaries with small tips and methods for achieving complete desolvation. Significant reduction of low-frequency and other noise was achieved in the image charge detector as well as determination of accurate methods for mass and charge measurement. Improved focusing and transmission efficiency was achieved via an aerodynamic lens. Suitable surfaces were also obtained including conductive diamond and fluorinated self-assembled monolayer (SAM) surfaces.

Traditional way to detect malicious software is based on signature matching. However, signature matching only detects known malicious software. In order to detect unknown malicious software, it is necessary to analyze the software for its impact on the system when the software is executed. In one approach, the software code can be statically analyzed for any malicious patterns. Another approach is to execute the program and determine the nature of the program dynamically. Since the execution of malicious code may have negative impact on the system, the code must be executed in a controlled environment. For that purpose, we have developed a sandbox to protect the system. Potential malicious behavior is intercepted by hooking Win32 system calls. Using the developed sandbox, we detect unknown virus using dynamic instruction sequences mining techniques. By collecting runtime instruction sequences in basic blocks, we extract instruction sequence patterns based on instruction associations. We build classification models with these patterns. By applying this classification model, we predict the nature of an unknown program. We compare our approach with several other approaches such as simple heuristics, NGram and static instruction sequences. We have also developed a method to identify a family of malicious software utilizing the system call trace. We construct a structural system call diagram from captured dynamic system call traces. We generate smart system call signature using profile hidden Markov model (PHMM) based on modularized system call block. Smart system call signature weakly identifies a family of malicious software.

Dengue virus (DENV) is the causative agent of a mosquito-transmitted disease mainly in tropical regions of the earth. Dengue is commonly diagnosed using polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA); however, these diagnostic methods both require complicated blood sample preparation, highly trained personnel, and centralized laboratory facilities, all of which are difficult to realize in many clinical settings where resources are limited. In the current study, a novel ultra-sensitive dendrimer-aptamer-based lateral flow assay (LFA) is designed to detect the presence of the DENV by detecting the envelope protein (E-Protein) of the DENV in phosphate-buffered saline (PBS) buffer and bovine serum albumin (BSA) sample. To achieve this, a “bioink”, a muti-handled streptavidin-dendrimer-aptamer conjugation is used to construct the modified test line in order to enhance the capturing efficiency of the signaling gold nanoparticle complexes on the test line. This work is the first time reported aptamer-based LFA of dengue virus detection. Our results show that the new LFA has a limit of detection of 24 pg/mL when tested using samples in PBS buffer (27 pg/mL in BSA solution), which is more sensitive that of a parallel ELISA test of 32 pg/mL and about ten-fold more sensitive than a conventional aptamer-based LFA. In addition, the new LFA shows that no non-specific binding with other E-protein in the flavivirus family and exhibits a long shelf-time for more than five weeks when stored in ambient conditions under subdued light. It can be concluded that the use of “bioink” -- a streptavidin-dendrimer-aptamer -- complex on the T-line can significantly enhance the detection sensitivity of the LFA assay. As a result, it is perceivable that the intrinsic portable, rapid, user-friendly, and cost-effective natures of LFAs in combination with the enhanced sensitivity due to the special fishnet-liked design will find broader applications for the LFAs as an effective and sufficiently sensitive diagnostic tool in many resources limited clinical settings.

jdelvall@upc.edu.pe / Acute respiratory infections are responsible for high morbi–mortality in Peruvian children. However, the etiological agents are poorly identified. This study, conducted during the pandemic outbreak of H1N1 influenza in 2009, aims to determine the main etiological agents responsible for acute respiratory infections in children from Lima, Peru. Nasopharyngeal swabs collected from 717 children with acute respiratory infections between January 2009 and December 2010 were analyzed by multiplex RT-PCR for 13 respiratory viruses: influenza A, B, and C virus; parainfluenza virus (PIV) 1, 2, 3, and 4; and human respiratory syncytial virus (RSV) A and B, among others. Samples were also tested with direct fluorescent-antibodies (DFA) for six respiratory viruses. RT-PCR and DFA detected respiratory viruses in 240 (33.5%) and 85 (11.9%) cases, respectively. The most common etiological agents were RSV-A (15.3%), followed by influenza A (4.6%), PIV-1 (3.6%), and PIV-2 (1.8%). The viruses identified by DFA corresponded to RSV (5.9%) and influenza A (1.8%). Therefore, respiratory syncytial viruses (RSV) were found to be the most common etiology of acute respiratory infections. The authors suggest that active surveillance be conducted to identify the causative agents and improve clinical management, especially in the context of possible circulation of pandemic viruses

The emergence of viruses in the family Paramyxoviridae, especially those such as Hendra virus and Nipah virus (NiV) that are zoonotic, highlighted the severity of disease that could be caused by infection with viruses belonging to this family. In addition to causing disease outbreaks, several newly discovered paramyxoviruses were found to have unique genetic features, which provoked renewed interest in the study of previously unclassified or uncharacterised viruses in this family. J-virus (JPV) was isolated from wild mice, in Queensland, Australia, in 1972, and has been suggested to be a natural respiratory pathogen of mice. Beilong virus (BeiPV), another paramyxovirus, was first isolated from human mesangial cells in Beijing, China, in 2003, and was subsequently detected in rat mesangial cells. Following initial characterisation, the genomes of JPV and BeiPV were found to contain two genes, SH and TM, not common to other paramyxoviruses, as well as an extended attachment protein gene. BeiPV has the largest genome in the family Paramyxoviridae, which is, in fact, larger than that of any other virus within the order Mononegavirales. The genetic material of paramyxoviruses is not amenable to manipulation via classical genetics; a reverse genetics approach was therefore employed to study the evolution and classification of JPV and BeiPV. Minireplicon systems utilising green fluorescent protein as a reporter were established for JPV, BeiPV and NiV, and were used to better assess the taxonomic status of JPV and BeiPV, and to determine the relationship between these viruses and henipaviruses, which also have exceptionally large genomes. These studies indicate that JPV and BeiPV are closely related and should be classified in the same genus and their replication and transcription machinery is different from that of the henipaviruses. / To gain an understanding of the biology of JPV and BeiPV, viral surface proteins from JPV were expressed and evaluated. Chimeric JPV virions containing recombinant surface proteins were generated and electron microscopy was used to determine the localisation of the proteins encoded by those JPV genes which are uncommon in other paramyxoviruses. Analysis of the attachment protein gene of JPV indicated that the virus was able to assemble an exceptionally large protein (156 kDa) into the virion structure, providing evidence in support of the hypothesis that JPV and BeiPV may represent an ancient lineage of viruses within the family Paramyxoviridae. In order to determine tissue tropism of JPV during experimental infection and to aid future work with a full-length JPV infectious clone, a real-time PCR assay for JPV was developed and assessed on tissues collected from mice infected with JPV. A multiplex microsphere assay for JPV and BeiPV was developed and used to analyse the seroprevalence of these viruses in Australian and Malaysian rodents. Although there is currently no evidence for disease caused by JPV or BeiPV, this does not preclude the emergence of a zoonotic rodent paramyxovirus related to these viruses. If this were to occur, the tools for virus detection and serological monitoring are now established.

Aleutian mink disease virus (AMDV) causes significant loss to the mink industry in Nova Scotia (NS). Contaminated water is a speculated virus source therefore my objective was to develop a method for detection of AMDV in water samples. Initially, the quality and quantity of AMDV DNA extracted by four commercial kits was evaluated by PCR. Next, viral capture by adsorption-elution filtration was tested using spiked water samples. The 1MDS filter method was superior or comparable to the MgCl2 filter method and outperformed the AlCl3 method. Lastly, 16 watershed samples from Southwest NS and 11 samples from two AMDV-infected ranches were tested. Five ranch samples from the two ranches tested positive. Three samples, containing more than 22 virus copies/µL after concentration were quantifiable. Pre-concentration, these samples contained 16,787 to 33,471 virus copies/mL. In conclusion, although further optimization is required, a method for the detection of AMDV in water samples was developed.

The polymerase chain reaction (PCR) is an extremely powerful tool for viral detection and screening because it can detect specific infectious agents with great sensitivity and specificity. It works by exponentially amplifying a target viral DNA sequence to high enough concentrations through the use of specific reagents and thermal cycling. It has surpassed culture based methods as the gold standard for viral detection because of the increased speed and sensitivity. Microfluidic approaches to PCR have focused on decreasing the time to thermally cycle, the volumes used for reactions, and they have also added upstream and downstream processes that are of benefit for on-chip viral detection. While these improvements have made great strides over commercially available products in terms of speed, cost, and integration, a major limitation that has yet to be explored is the throughput associated with running PCR. Since each PCR reaction relies on primers with a unique annealing temperature to detect specific viral DNA, only a single virus can be screened for at a time. The device presented here uses two infrared laser diodes that are driven identically by the same laser driver to independently thermally cycle two chambers on the same microfluidic chip. Different temperatures are achieved in the two chambers by modulating the radiation reaching one of those chambers with an optical shutter. Closed loop temperature feedback in both chambers is done with a Labview program and thermocouples embedded in the polymer chip. This allows for accurate temperature measurement without inhibiting the reaction. To demonstrate the capabilities of this device, two different reactions were simultaneously amplified successfully on the same device that have annealing temperatures that differ by 15°C.