A novel tracer, glucose oxidase-functionalized nanocomposite, was designed to label the signal antibodies for ultrasensitive multiplexed measurement of tumor markers using a disposable immunosensor array. The immunosensor array was constructed by coating layer-by-layer colloidal Prussian blue (PB), gold nanoparticles, and capture antibodies on screen-printed carbon electrodes. The preparation of glucose oxidase-functionalized nanocomposites and the labeling of antibody were performed by one-pot assembly of glucose oxidase and antibody on gold nanoparticles attached carbon nanotubes. The PB immobilized on immunosensor surface acted as a mediator to catalyze the reduction of H2O2 produced in the enzymatic cycle. Both the high-content glucose oxidase and carbon nanotubes in the tracer amplified the detectable signal for the sandwich-type immunoassay. Using carcinoembryonic antigen and alpha-fetoprotein as model analytes, the simultaneous multiplexed immunoassay method using the immunosensor array and the designed tracer showed linear ranges of 3 orders of magnitude with the detection limits down to 1.4 and 2.2 pg/mL, respectively. The assay results of serum samples with the proposed method were in an acceptable agreement with the reference values. The dual signal amplification of glucose oxidase-functionalized nanocomposites provided a promising ultrasensitive simultaneous multiplexed immunoassay approach for clinical applications.
Porcine circovirus type 3 (PCV3) is an emerging porcine circovirus that has been associated with porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, reproductive failure, cardiac pathologies, and multisystemic inflammation in piglets and sows. Many aspects of PCV3 infection biology and pathogenesis, however, remain unknown. Here, we used a PCV3 virus stock from the rescue of an infectious PCV3 DNA clone to intranasally inoculate 4- and 8-week-old specific-pathogen-free piglets for evaluation of PCV3 pathogenesis. For 4-week-old piglets, typical clinical signs resembling those of PDNS-like disease were observed when piglets were inoculated with PCV3 alone or PCV3 combined with immunostimulation by keyhole limpet hemocyanin, with a mortality of 40% (2/5) for both types of inoculated piglets during a 28-day observation period postinoculation. Both types of inoculated piglets showed similar progressive increases in viral loads in the sera and had seroconverted to PCV3 capsid antibody after inoculation. Pathological lesions and PCV3-specific antigen were detected in various tissues and organs, including the lung, heart, kidney, lymph nodes, spleen, liver, and small intestine, in both types of inoculated piglets. The levels of proinflammatory cytokines and chemokines, including interleukin 1 beta (IL-1β), IL-6, IL-23α, gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and chemokine ligand 5 (CCL5), were significantly upregulated in both groups of inoculated piglets. Eight-week-old piglets also exhibited a similar PDNS-like disease but without death after PCV3 inoculation, as evidenced by pathological lesions and PCV3 antigen in various tissues and organs. These results show for the first time successful reproduction of PDNS-like disease by PCV3 infection and further provide significant information regarding the pathogenesis of PCV3 in piglets. IMPORTANCE Porcine circovirus type 3 (PCV3), an emerging porcine circovirus, is considered the cause of porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs and other systemic diseases in piglets and sows. To evaluate the pathogenesis of PCV3 infection in vivo, we used a PCV3 virus stock from the rescue of an infectious PCV3 DNA clone to intranasally inoculate 4- and 8-week-old specific-pathogen-free piglets and demonstrated successful reproduction of PDNS-like disease in animals that were inoculated with PCV3 alone or PCV3 combined with immunostimulation by keyhole limpet hemocyanin. Both 4- and 8-week-old PCV3-inoculated piglets showed similar increases in viral loads in the sera and had seroconverted to PCV3 capsid antibody. Pathological lesions and PCV3-specific antigen were detected in various tissues and organs, while numerous proinflammatory cytokines and chemokines in the sera were significantly upregulated after PCV3 inoculation. These results will provide significant information regarding the pathogenesis of PCV3 in piglets.
Hendra virus (HeV) is a recently emerged zoonotic paramyxovirus that can cause a severe and often fatal disease in horses and humans. HeV is categorized as a biosafety level 4 agent, which has made the development of animal models and testing of potential therapeutics and vaccines challenging. Infection of African Green monkeys (AGMs) with HeV was recently demonstrated and disease mirrored fatal HeV infection in humans, manifesting as a multisystemic vasculitis with widespread virus replication in vascular tissues and severe pathologic manifestations in the lung, spleen and brain. Here, we demonstrate that m102.4, a potent HeV neutralizing human monoclonal antibody (hmAb), can protect AGMs from disease post infection (p.i.) with HeV. Fourteen AGMs were challenged intratracheally with a lethal dose of HeV and twelve subjects were infused twice with a 100 mg dose of m102.4 beginning at either 10 hr, 24 hr or 72 hr p.i. and again approximately 48 hrs later. The presence of viral RNA, infectious virus and HeV-specific immune responses demonstrated that all subjects were infected following challenge. All twelve AGMs that received m102.4 survived infection; whereas the untreated control subjects succumbed to disease on day 8 p.i.. Animals in the 72 hr treatment group exhibited neurological signs of disease but all animals started to recover by day 16 p.i.. These results represent successful post-exposure in vivo efficacy by an investigational drug against HeV and highlight the potential impact a hmAb can have on human disease.
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