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In this work, we report a 5-min magnetic particle spectroscopy (MPS)-based bioassay strategy. In our approach, surface-functionalized magnetic nanoparticles are incubated with target analytes at 37 °C with agitation for 3 min, and the MPS reading is then taken at the fifth minute. We prove the feasibility of 5 min ultrafast detection of SARS-CoV-2 spike protein with a detection limit below 5 nM (0.2 pmol). Our proposed 5-min bioassay strategy may be applied to reduce the assay time for other liquid-phase, volumetric biosensors such as NMR, quantum dots, fluorescent biosensors, etc.

With the ongoing global pandemic of coronavirus disease 2019 (COVID-19), there is an increasing quest for more accessible, easy-to-use, rapid, inexpensive, and high-accuracy diagnostic tools. Traditional disease diagnostic methods such as qRT-PCR (quantitative reverse transcription-PCR) and ELISA (enzyme-linked immunosorbent assay) require multiple steps, trained technicians, and long turnaround time that may worsen the disease surveillance and pandemic control. In sight of this situation, a rapid, one-step, easy-to-use, and high-accuracy diagnostic platform will be valuable for future epidemic control, especially for regions with scarce medical resources. Herein, we report a magnetic particle spectroscopy (MPS) platform for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biomarkers: spike and nucleocapsid proteins. This technique monitors the dynamic magnetic responses of magnetic nanoparticles (MNPs) and uses their higher harmonics as a measure of the nanoparticles' binding states. By anchoring polyclonal antibodies (pAbs) onto MNP surfaces, these nanoparticles function as nanoprobes to specifically bind to target analytes (SARS-CoV-2 spike and nucleocapsid proteins in this work) and form nanoparticle clusters. This binding event causes detectable changes in higher harmonics and allows for quantitative and qualitative detection of target analytes in the liquid phase. We have achieved detection limits of 1.56 nM (equivalent to 125 fmole) and 12.5 nM (equivalent to 1 pmole) for detecting SARS-CoV-2 spike and nucleocapsid proteins, respectively. This MPS platform combined with the one-step, wash-free, nanoparticle clustering-based assay method is intrinsically versatile and allows for the detection of a variety of other disease biomarkers by simply changing the surface functional groups on MNPs.

In recent years, magnetic particle spectroscopy (MPS) has become a highly sensitive and versatile sensing technique for quantitative bioassays. It relies on the dynamic magnetic responses of magnetic nanoparticles (MNPs) for the detection of target analytes in the liquid phase. There are many research studies reporting the application of MPS for detecting a variety of analytes including viruses, toxins, nucleic acids, and so forth. Herein, we report a modified version of the MPS platform with the addition of a one-stage lock-in design to remove the feedthrough signals induced by external driving magnetic fields, thus capturing only MNP responses for improved system sensitivity. This one-stage lock-in MPS system is able to detect as low as 781 ng multi-core Nanomag50 iron oxide MNPs (micromod Partikeltechnologie GmbH) and 78 ng single-core SHB30 iron oxide MNPs (Ocean NanoTech). We first demonstrated the performance of this MPS system for bioassay-related applications. Using the SARS-CoV-2 spike protein as a model, we have achieved a detection limit of 125 nM (equal to 5 pmole) for detecting spike protein molecules in the liquid phase. In addition, using a streptavidin-biotin binding system as a proof-of-concept, we show that these single-core SHB30 MNPs can be used for Brownian relaxation-based bioassays while the multi-core Nanomag50 cannot be used. The effects of MNP amount on the concentration-dependent response profiles for detecting streptavidin were also investigated. Results show that by using a lower concentration/ amount of MNPs, concentration-response curves shift to a lower concentration/amount of target analytes. This lower concentration-response indicates the possibility of improved bioassay sensitivities by using lower amounts of MNPs.

Blastocyst complementation combined with gene editing is an emerging approach in the field of regenerative medicine that could potentially solve the worldwide problem of organ shortages for transplantation. In theory, blastocyst complementation can generate fully functional human organs or tissues, grown within genetically engineered livestock animals. Targeted deletion of a specific gene(s) using gene editing to cause deficiencies in organ development can open a niche for human stem cells to occupy, thus generating human tissues. Within this review, we will focus on the pancreas, liver, heart, kidney, lung, and skeletal muscle, as well as cells of the immune and nervous systems. Within each of these organ systems, we identify and discuss (i) the common causes of organ failure; (ii) the current state of regenerative therapies; and (iii) the candidate genes to knockout and enable specific exogenous organ development via the use of blastocyst complementation. We also highlight some of the current barriers limiting the success of blastocyst complementation.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to mutate, causing disruptive PRRS outbreaks in farms that lead to reproductive failure and respiratory disease-associated mortality. We present four new PRRSV type 2 variants in the United States belonging to four distinct orf5 sublineages within lineage 1.

The magnitude of SARS-CoV-2–specific T cell responses correlates inversely with human disease severity, suggesting T cell involvement in primary control. Whereas many COVID-19 vaccines focus on establishing humoral immunity to viral spike protein, vaccine-elicited T cell immunity may bolster durable protection or cross-reactivity with viral variants. To better enable mechanistic and vaccination studies in mice, we identified a dominant CD8 T cell SARS-CoV-2 nucleoprotein epitope. Infection of human ACE2 transgenic mice with SARS-CoV-2 elicited robust responses to H2-Db/N219-227, and 40% of HLA-A*02+ COVID-19 PBMC samples isolated from hospitalized patients responded to this peptide in culture. In mice, i.m. prime-boost nucleoprotein vaccination with heterologous vectors favored systemic CD8 T cell responses, whereas intranasal boosting favored respiratory immunity. In contrast, a single i.v. immunization with recombinant adenovirus established robust CD8 T cell memory both systemically and in the respiratory mucosa.

Zika virus (ZIKV) exhibits a tropism for brain tumor cells and has been used as an oncolytic virus to target brain tumors in mice with modest effects on extending median survival. Recent studies have highlighted the potential for combining virotherapy and immunotherapy to target cancer. We postulated that ZIKV could be used as an adjuvant to enhance the long-term survival of mice with malignant glioblastoma and generate memory T-cells capable of providing long-term immunity against cancer remission. To test this hypothesis mice bearing malignant intracranial GL261 tumors were subcutaneously vaccinated with irradiated GL261 cells previously infected with the ZIKV. Mice also received intracranial injections of live ZIKV, irradiated ZIKV, or irradiated GL261 cells previously infected with ZIKV. Long-term survivors were rechallenged with a second intracranial tumor to examine their immune response and look for the establishment of protective memory T-cells. Mice with subcutaneous vaccination plus intracranial irradiated ZIKV or intracranial irradiated GL261 cells previously infected with ZIKV exhibited the greatest extensions to overall survival. Flow cytometry analysis of immune cells within the brains of long-term surviving mice after tumor rechallenge revealed an upregulation in the levels of T-cells, including CD4+ and tissue-resident memory CD4+ T-cells, in comparison to long-term survivors that were mock-rechallenged, and in comparison to naïve untreated mice challenged with intracranial gliomas. These results suggest that ZIKV can serve as an adjuvant to subcutaneous tumor vaccines that enhance long-term survival and generate protective tissue-resident memory CD4+ T-cells.

In recent years a number of new nanotechnology based platforms have been developed for detection of wide variety of targets including infectious agents, protein biomarkers, nucleic acids, drugs, and cancer cells. Nanomaterials such as magnetic nanoparticles, quantum dots, carbon nanotubes, nanowires, and nanosensors like giant magnetoresistance (GMR) sensors are used to quantitatively detect biomolecules with, experimentally, relatively good accuracy. There has been a growing interest in the use of magnetic fields in biosensing applications. Because biological samples have no ferromagnetic property and therefore there is no interference with complex sample matrix, detection of infectious agents from minimally processed samples is possible. Here, we provide a brief overview of the recent emergence of nanotechnology-based techniques for the detection and monitoring of foodborne diseases. In addition, the potential applications and future perspectives of nanotechnology on food safety are discussed. Ultimately, the review is expected to stimulate and provide directions to the development and application of nanotechnology-based tests for the early detection, and eventual control of foodborne diseases.

Influenza A virus (IAV) is a common respiratory pathogen infecting many hosts including humans, pigs (swine influenza virus or SIV), and birds (avian influenza virus or AIV). Monitoring swine and avian influenza viruses in the wild, farms, and live poultry markets is of great significance for human and veterinary public health. A portable, sensitive, and quantitative immunoassay device will be of high demand especially in the rural and resource-limited areas. We report herein our Z-Lab point-of-care (POC) device for sensitive and specific detection of swine influenza viruses with minimum sample handling and laboratory skill requirements. In the present study, a portable and quantitative immunoassay platform based on giant magnetoresistive (GMR) technology is used for the detection of IAV nucleoprotein (NP) and purified H3N2v. Z-Lab displays quantitative results in less than 10 min with sensitivities down to 15 ng/mL and 125 TCID50/mL for IAV nucleoprotein and purified H3N2v, respectively. This platform al...

Understanding the immune responses against Porcine epidemic diarrhea virus (PEDV) is important to prevent infection and to design control strategies. We evaluated both systemic and mucosal immune responses to PEDV in pigs and assessed if prior exposure to virus protects against re-infection. Three-week-old pigs were infected with PEDV and immune response in blood, intestine, and mesenteric lymph node (MLN) was evaluated. At 30 dpi, virus exposed pigs were challenged with a field isolate of PEDV and immune response at 5 d post challenge was evaluated. We found that PEDV RNA persists in the intestine even after fecal shedding of the virus was stopped at 28 dpi and pigs previously exposed to PEDV are protected from virus shedding after re-infection. PEDV infection induced both humoral and cell mediated immune response with an increase in PEDV specific IgA and IgG antibodies in intestine and serum. Flow cytometry analysis showed a significantly higher frequency of B cells and lower frequency of T cells at 4 dpi. The frequency of CD4/CD8 double positive (DP) memory T cells was significantly increased in the MLN of challenged animals. These studies may provide further insights into understanding the mucosal immune response to PEDV and its role in protection against disease.

Indirect transmission of porcine epidemic diarrhea virus (PEDV) ensues when susceptible animals contact PEDV-contaminated fomite materials. Although the survival of PEDV under various pHs and temperatures has been studied, virus stability on different fomite surfaces under varying temperature conditions has not been explored. Hence, we evaluated the survival of PEDV on inanimate objects routinely used on swine farms such as styrofoam, rubber, plastic, coveralls, and other equipment. The titer of infectious PEDV at 4 °C decreased by only 1 to 2 log during the first 5 days, and the virus was recoverable for up to 15 days on Styrofoam, aluminum, Tyvek® coverall, cloth, and plastic. However, viral titers decreased precipitously when stored at room temperature; no virus was detectable after one day on all materials tested. A more sensitive immunoplaque assay was able to detect virus from Styrofoam, metal, and plastic at 20 days post application, representing a 3-log loss of input virus on fomite materials. Recovery of infectious PEDV from Tyvek® coverall and rubber was above detection limit at 20 days. Our findings indicate that the type of fomite material and temperatures impact PEDV stability, which is important in understanding the nuances of indirect transmission and epidemiology of PEDV.

We have developed a simple and sensitive method for the detection of influenza A virus based on giant magnetoresistance (GMR) biosensor. This assay employs monoclonal antibodies to viral nucleoprotein (NP) in combination with magnetic nanoparticles (MNPs). Presence of influenza virus allows the binding of MNPs to the GMR sensor and the binding is proportional to the concentration of virus. Binding of MNPs onto the GMR sensor causes change in the resistance of sensor, which is measured in a real time electrical readout. GMR biosensor detected as low as 1.5 × 10(2) TCID50/mL virus and the signal intensity increased with increasing concentration of virus up to 1.0 × 10(5) TCID50/mL. This study showed that the GMR biosensor assay is relevant for diagnostic application since the virus concentration in nasal samples of influenza virus infected swine was reported to be in the range of 10(3) to 10(5) TCID50/mL.

Japanese encephalitis virus(JEV), a single-stranded positive-sense RNA virus of the familyFlaviviridae, is the major cause of paediatric encephalitis in Asia. The high incidence of subclinical infections in Japanese encephalitis-endemic areas and subsequent evasion of encephalitis points to the development of immune responses against JEV. Humoral responses play a central role in protection against JEV; however, cell-mediated immune responses contributing to this end are not fully understood. The structural envelope (E) protein, the major inducer of neutralizing antibodies, is a poor target for T cells in natural JEV infections. The extent to which JEV non-structural proteins are targeted by T cells in subclinically infected healthy children would help to elucidate the role of cell-mediated immunity in protection against JEV as well as other flaviviral infections. The property of the Tat peptide ofHuman immunodeficiency virusto transduce proteins across cell membranes, facilitating intracellular protein delivery following exogenous addition to cultured cells, prompted us to express the four largest proteins of JEV, comprising 71 % of the JEV genome coding sequence, as Tat fusions for enumerating the frequencies of virus-specific CD4+and CD8+T cells in JEV-immune donors. At least two epitopes recognized by distinct HLA alleles were found on each of the non-structural proteins, with dominant antiviral Th1 T cell responses to the NS3 protein in nearly 96 % of the cohort. The data presented here show that non-structural proteins are frequently targeted by T cells in natural JEV infections and may be efficacious supplements for the predominantly antibody-eliciting E-based JEV vaccines.

We demonstrate the presence of nonstructural protein 1 (NS1)-specific antibodies in a significant proportion of convalescent-phase human serum samples obtained from a cohort in an area where Japanese encephalitis virus (JEV) is endemic. Sera containing antibodies to NS1 but not those with antibodies to other JEV proteins, such as envelope, brought about complement-mediated lysis of JEV-infected BHK-21 cells. Target cells infected with a recombinant poxvirus expressing JEV NS1 on the cell surface confirmed the NS1 specificity of cytolytic antibodies. Mouse anti-NS1 cytolytic sera caused a complement-dependent reduction in virus output from infected human cells, demonstrating their important role in viral control. Antibodies elicited by JEV NS1 did not cross lyse West Nile virus- or dengue virus-infected cells despite immunoprecipitating the NS1 proteins of these related flaviviruses. Additionally, JEV NS1 failed to bind complement factor H, in contrast to NS1 of West Nile virus, suggesting that the NS1 proteins of different flaviviruses have distinctly different mechanisms for interacting with the host. Our results also point to an important role for JEV NS1-specific human immune responses in protection against JE and provide a strong case for inclusion of the NS1 protein in next generation of JEV vaccines.

Interferons (IFNs) have been shown to inhibit influenza A virus (IAV) replication and play an essential role in controlling viral infection. Here we studied the kinetics and magnitude of induction of type I and type III IFN transcripts by primary porcine airway epithelial cells (pAECs) in response to swine and human origin IAV. We observed that swine influenza viruses (SIV) replicate more efficiently than the human pandemic influenza A/California/2009 (pH1N1 CA/09) in pAECs. Interestingly, we also found significant difference in kinetics of IFN-β, IFN-λ1 and IFN-λ3 gene expression by these viruses. While there was delay of up to 12 hours post infection (h p.i.) in induction of IFN genes in pAECs infected with swine IAV A/Sw/Illinois/2008 (H1N1 IL/08), human pH1N1 CA/09 rapidly induced IFN-β, IFN-λ1 and IFN-λ3 gene expression as early as 4 h p.i. However, the magnitude of IFN-β and IFN-λ3 induction at 24 h p.i. was not significantly different between the viral strains tested. Additionally, we found that swine H1N1 IL/08 was less sensitive to dsRNA induced antiviral response compared to human pH1N1 CA/09. Our data suggest that the human and swine IAVs differ in their ability to induce and respond to type I and type III interferons in swine cells. Swine origin IAV may have adapted to the pig host by subverting innate antiviral responses to viral infection.