Tetracore (United States)

ABSTRACTPorcine hemagglutinating encephalomyelitis virus (PHEV) is a betacoronavirus that causes vomiting and wasting disease and/or encephalomyelitis in suckling pigs. This study characterized PHEV infection, pathogenesis, and immune response in cesarean-derived, colostrum-deprived (CDCD) neonatal pigs. Infected animals developed mild respiratory, enteric, and neurological clinical signs between 2 to 13 days postoronasal inoculation (dpi). PHEV did not produce viremia, but virus shedding was detected in nasal secretions (1 to 10 dpi) and feces (2 to 7 dpi) by reverse transcriptase quantitative PCR (RT-qPCR). Viral RNA was detected in all tissues except liver, but the detection rate and RT-qPCR threshold cycle (CT) values decreased over time. The highest concentration of virus was detected in inoculated piglets necropsied at 5 dpi in turbinate and trachea, followed by tonsils, lungs, tracheobronchial lymph nodes, and stomach. The most representative microscopic lesions were gastritis lymphoplasmacytic, moderate, multifocal, with perivasculitis, and neuritis with ganglia degeneration. A moderate inflammatory response, characterized by increased levels of interferon alpha (IFN-α) in plasma (5 dpi) and infiltration of T lymphocytes and macrophages were also observed. Increased plasma levels of interleukin-8 (IL-8) were detected at 10 and 15 dpi, coinciding with the progressive resolution of the infection. Moreover, a robust antibody response was detected by 10 dpi. An ex vivo air-liquid CDCD-derived porcine respiratory cells culture (ALI-PRECs) system showed virus replication in ALI-PRECs and cytopathic changes and disruption of ciliated columnar epithelia, thereby confirming the tracheal epithelia as a primary site of infection for PHEV.IMPORTANCE Among the ∼46 virus species in the family Coronaviridae, many of which are important pathogens of humans and 6 of which are commonly found in pigs, porcine hemagglutinating encephalomyelitis remains one of the least researched. The present study provided a comprehensive characterization of the PHEV infection process and immune responses using CDCD neonatal pigs. Moreover, we used an ex vivo ALI-PRECs system resembling the epithelial lining of the tracheobronchial region of the porcine respiratory tract to demonstrate that the upper respiratory tract is a primary site of PHEV infection. This study provides a platform for further multidisciplinary studies of coronavirus infections.

Single-domain antibodies (sdAb), recombinantly produced variable heavy domains derived from the unconventional heavy chain antibodies found in camelids, provide stable, well-expressed binding elements with excellent affinity that can be tailored for specific applications through protein engineering. Complex matrices, such as plasma and serum, can dramatically reduce assay sensitivity. Thus, to achieve highly sensitive detection in complex matrices a highly efficient assay is essential. We produced sdAb as genetically linked dimers, and trimers, each including SpyTag at their C-terminus. The constructs were immobilized onto dyed magnetic microspheres to which SpyCatcher had been coupled and characterized in terms of their performance as capture reagents in sandwich assays. Initial tests on the ability of oriented monomer, dimer, and trimer captures to improve detection versus unoriented constructs in an assay for staphylococcal enterotoxin B spiked into buffer showed the oriented dimer format provided the best sensitivity while offering robust protein production. Thus, this format was utilized to improve a sdAb-based assay for the detection of dengue virus (DENV) nonstructural protein 1 (NS1) in serum. Detection of NS1 from each of the four DENV serotypes spiked into 50% normal human serum was increased by at least a factor of 5 when using the oriented dimer capture. We then demonstrated the potential of using the oriented dimer capture to improve detection of NS1 in clinical samples. This general method should enhance the utility of sdAb incorporated into any diagnostic assay, including those for high consequence pathogens.

Abstract Many cancers overexpress FASN which catalyzes the final synthetic step of de novo fatty acid synthesis pathway. FASN has been considered as a potential drug target for several decades now. Many publications provide evidence of the elevated FASN with aggressive tumors, but still, there are very few inhibitors of FASN that have successfully been utilized as therapeutics in the clinical arena. An accurate measurement of FASN in association with tumor-specific markers in solid tumors and serum may help to propel forward the therapeutic significance of FASN inhibitors. With this objective in mind, we are currently working on the development of a multiplex assay for detection and quantitation of FASN and Cripto-1 in serum samples. We have optimized a magnetic fluorescent bead based multiplex sandwich immunoassay for detection and quantitation of FASN and Cripto-1 (CR-1) in human serum and plasma samples. We selected Cripto-1 to pair with FASN because it is an EGF-like domain carrying growth factor associated with a number of different types of human carcinomas. We use two different monoclonal antibodies for FASN and one for CR-1 as capture antibodies covalently coupled to magnetic fluorescent beads and a mixture of two biotin-labeled detection antibodies in this assay. The streptavidin-phycoerythrin (SAPE) conjugate is used to report the positive capture of a target in this assay. We have also included various internal controls in this assay to monitor the assay performance and assure quality of the reagents used in each well. The dynamic range of this assay was determined to be 100pg/mL to 100 ng/mL using recombinant FASN and Cripto-1 from commercial sources. The R2 values were 0.98 and above for each of the analytes and recovery of each standard were between 90 % and 125%. We spiked recombinant FASN and Cripto-1 in normal human serum and plasma at different concentrations and spike recovery was between 70 % and 130 %. We further evaluated this assay with some commercially obtained clinical samples. This presentation includes detailed results of this study. Citation Format: Neeraja Venkateswaran, William M. Nelson. A novel multiplex assay for detection and quantitation of fatty acid synthase (FASN) in serum or plasma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-248.

Influenza A virus (IAV) surveillance using pre-weaning oral fluid samples from litters of piglets was evaluated in four ˜12500 sow and IAV-vaccinated, breeding herds. Oral fluid samples were collected from 600litters and serum samples from their dams at weaning. Litter oral fluid samples were tested for IAV by virus isolation, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), RT-PCR subtyping and sequencing. Commercial nucleoprotein (NP) enzyme-linked immunosorbent assay (ELISA) kits and NP isotype-specific assays (IgM, IgA and IgG) were used to characterize NP antibody in litter oral fluid and sow serum. All litter oral fluid specimens (n=600) were negative by virus isolation. Twenty-five oral fluid samples (25/600=4.2%) were qRT-PCR positive based on screening (Laboratory 1) and confirmatory testing (Laboratory 2). No hemagglutinin (HA) and neuraminidase (NA) gene sequences were obtained, but matrix (M) gene sequences were obtained for all qRT-PCR-positive samples submitted for sequencing (n=18). Genetic analysis revealed that all M genes sequences were identical (GenBank accession no. KF487544) and belonged to the triple reassortant influenza A virus M gene (TRIG M) previously identified in swine. The proportion of IgM- and IgA-positive samples was significantly higher in sow serum and litter oral fluid samples, respectively (P<0.01). Consistent with the extensive use of IAV vaccine, no difference was detected in the proportion of IgG- and blocking ELISA-positive sow serum and litter oral fluids. This study supported the use of oral fluid sampling as a means of conducting IAV surveillance in pig populations and demonstrated the inapparent circulation of IAV in piglets. Future work on IAV oral fluid diagnostics should focus on improved procedures for virus isolation, subtyping and sequencing of HA and NA genes. The role of antibody in IAV surveillance remains to be elucidated, but longitudinal assessment of specific antibody has the potential to provide information regarding patterns of infection, vaccination status and herd immunity.

Detection of proteinaceous toxins in complex heterogeneous mixtures requires highly specific and sensitive methods. Multiplex technology employing multiple antibodies that recognize different epitopes on a toxin provides built-in confirmatory analysis as part of the initial screen and thereby increases the reliability associated with both presumptive positive and negative results. Polyclonal and monoclonal antibodies were obtained for abrin, botulinum toxins, ricin, and Staphylococcus enterotoxins A, B, and C (SEA, SEB, and SEC). Food samples were spiked with the toxins either individually or mixed and analyzed following 40-fold dilution. Abrin, botulinum toxin A complex, ricin, and SEB displayed limits of detection in the original food samples ranging from 0.03 to 1.3 microg/mL, from 0.03 to 0.07 microg/mL, from 0.01 to 0.1 microg/mL, and from <0.01 to 0.03 microg/mL, respectively. Redundancy, that is, multiple antibodies for each toxin, some recognizing different epitopes or displaying different binding affinities, provided a "fingerprint" for the presence of the toxins and built-in confirmation, thus reducing the likelihood of false-positive and false-negative results. Inclusion of internal controls, including a unique protein, helped control for variations in dilution. Paramagnetic microspheres facilitated the detection of analyte in foods containing particulate matter incompatible with the use of filter plates normally used in the wash steps of assays employing standard polystyrene microspheres.

BackgroundMutations in the mitochondrial genome (mtgenome) have been associated with cancer and many other disorders. These mutations can be point mutations or deletions, or admixtures (heteroplasmy). The detection of mtDNA mutations in body fluids using resequencing microarrays, which are more sensitive than other sequencing methods, could provide a strategy to measure mutation loads in remote anatomical sites.MethodsWe determined the mtDNA mutation load in the entire mitochondrial genome of 26 individuals with different early stage cancers (lung, bladder, kidney) and 12 heavy smokers without cancer. MtDNA was sequenced from three matched specimens (blood, tumor and body fluid) from each cancer patient and two matched specimens (blood and sputum) from smokers without cancer. The inherited wildtype sequence in the blood was compared to the sequences present in the tumor and body fluid, detected using the Affymetrix Genechip® Human Mitochondrial Resequencing Array 1.0 and supplemented by capillary sequencing for noncoding region.ResultsUsing this high-throughput method, 75% of the tumors were found to contain mtDNA mutations, higher than in our previous studies, and 36% of the body fluids from these cancer patients contained mtDNA mutations. Most of the mutations detected were heteroplasmic. A statistically significantly higher heteroplasmy rate occurred in tumor specimens when compared to both body fluid of cancer patients and sputum of controls, and in patient blood compared to blood of controls. Only 2 of the 12 sputum specimens from heavy smokers without cancer (17%) contained mtDNA mutations. Although patient mutations were spread throughout the mtDNA genome in the lung, bladder and kidney series, a statistically significant elevation of tRNA and ND complex mutations was detected in tumors.ConclusionOur findings indicate comprehensive mtDNA resequencing can be a high-throughput tool for detecting mutations in clinical samples with potential applications for cancer detection, but it is unclear the biological relevance of these detected mitochondrial mutations. Whether the detection of tumor-specific mtDNA mutations in body fluidsy this method will be useful for diagnosis and monitoring applications requires further investigation.

Ricin is a toxic ribosome inactivating protein (RIP-II) present in beans of the castor plant, Ricinus communis. Its potential as a biodefense threat has made the rapid, sensitive detection of ricin in food important to the U.S. Food and Drug Administration. Samples of juice, dairy products, soda, vegetables, bakery products, chocolate, and condiments were spiked with varying concentrations of ricin and analyzed using a 96-well format, electrochemiluminescence (ECL) immunoassay. Assay configurations included the use of a monoclonal capture antibody coupled with either a polyclonal or monoclonal detector antibody. The samples and detector antibodies were either added sequentially or in combination during the capture step. Using the polyclonal antibody, 0.04 ng/mL ricin was detected in analytical samples prepared from several beverages. By simultaneously incubating the sample with detector antibody, it was possible to decrease the assay time to a single 20 min incubation step with a limit of detection <10 ng/mL. Assays run according to this single incubation step exhibited a hook effect (decrease in signal at high concentrations of ricin), but because of the large signal-to-noise ratio associated with the ECL assay, the response remained above background and detectable. Thus, the ECL assay was uniquely suited for the screening of samples for ricin.

This report further documents the critical importance of identifying super-shedders, since they contribute billions of MAP CFU daily to the environment, serving to disseminate MAP to susceptible cattle. In many herds, this results in significant numbers of culture-positive fecal samples through passive shedding. The frequency of super-shedders among culture-positive cattle in infected herds is unknown. However, our preliminary investigations have shown that each of three current herds has at least one super-shedder cow, with five super-shedder cows having been identified among the 7.1% ( 41 of 575) of culture-positive cattle in the herds. Our preliminary estimates are that 10% of heavy shedders (or about 2 to 3% of all culture-positive cattle at a single time-point) may be super-shedders, excreting &gt;10 billion CFU MAP per cow per day.