Universal primer multiplex PCR assay for detection and genotyping of porcine astroviruses.

Porcine astroviruses (PAstV) are members of the family Astroviridae, Mamastravirus genus and have been identified to have five genotypes (PAstV1-5). These viruses are highly prevalent in pigs and can cause enteric disease as well as neurological or respiratory symptoms depending on their genotypes. At present, the epidemiological impacts of some PAstV genotypes on pigs are largely unknown and hence continuously monitoring of these PAstVs may be needed.

Spike-specific T cells are enriched in breastmilk following SARS-CoV-2 mRNA vaccination.

Human breastmilk is rich in T cells; however, their specificity and function are largely unknown. We compared the phenotype, diversity, and antigen specificity of T cells in breastmilk and peripheral blood of lactating individuals who received SARS-CoV-2 messenger RNA (mRNA) vaccination. Relative to blood, breastmilk contained higher frequencies of T effector and central memory populations that expressed mucosal-homing markers.

Multiplex gel-based PCR assay for the simultaneous detection of 5 genotypes of porcine astroviruses.

Porcine astrovirus (PAstV) has been associated experimentally with diarrhea in piglets, but much more knowledge is needed about this virus. PAstV has high genetic variability, and 5 genotypes have been identified, namely PAstV1-5. To obtain information on the epidemiology of PAstV, we established a multiplex PAstV PCR assay to detect and differentiate the 5 PAstV genotypes simultaneously.

Persistent Nonviral Plasmid Vector in Nasal Tissues Causes False-Positive SARS-CoV-2 Diagnostic Nucleic Acid Tests.

Biomedical personnel can become contaminated with nonhazardous reagents used in the laboratory. We describe molecular studies performed on nasal secretions collected longitudinally from asymptomatic laboratory coworkers to determine if they were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) circulating in the community or with SARS-CoV-2 DNA from a plasmid vector. Participants enrolled in a prospective study of incident SARS-CoV-2 infection had nasal swabs collected aseptically by study staff at enrollment, followed by weekly self-collection of anterior nasal swabs.

Spike-specific T cells are enriched in breastmilk following SARS-CoV-2 mRNA vaccination.

Unlabelled: Human breastmilk is rich in T cells; however, their specificity and function are largely unknown. We compared the phenotype, diversity, and antigen specificity of T cells in the breastmilk and peripheral blood of lactating individuals who received SARS-CoV-2 mRNA vaccination. Relative to blood, breastmilk contained higher frequencies of T effector and central memory populations that expressed mucosal-homing markers.

Low rate of SARS-CoV-2 incident infection identified by weekly screening PCR in a prospective year-long cohort study.

Background: Asymptomatic and pre-symptomatic SARS-CoV-2 infections may contribute to ongoing community transmission, however, the benefit of routine screening of asymptomatic individuals in low-risk populations is unclear.

Methods: To identify SARS-CoV-2 infections 553 seronegative individuals were prospectively followed for 52 weeks. From 4/2020-7/2021, participants submitted weekly self-collected nasal swabs for rtPCR and completed symptom and exposure surveys.

Angiotensin II receptor I auto-antibodies following SARS-CoV-2 infection.

Background: Coronavirus disease 2019 (COVID-19) is associated with endothelial activation and coagulopathy, which may be related to pre-existing or infection-induced pro-thrombotic autoantibodies such as those targeting angiotensin II type I receptor (AT1R-Ab).

Methods: We compared prevalence and levels of AT1R-Ab in COVID-19 cases with mild or severe disease to age and sex matched negative controls utilizing multivariate logistic and quantile regression adjusted for comorbidities including hypertension, diabetes, and heart disease.

Results: There were trends toward increased prevalence (50% vs.

Novel universal primer-pentaplex PCR assay based on chimeric primers for simultaneous detection of five common pig viruses associated with diarrhea.

Viral pathogens associated with diarrhea in pigs include porcine circovirus 2 (PCV2), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (RVA) and C (RVC) among others. In this study, a novel universal primer-based pentaplex PCR (UP-M-PCR) assay was developed for simultaneous detection and differentiation of these five viruses. The assay uses a short-cycle multiplex amplification by chimeric primers (CP), which are virus specific, with a tail added at the 5' end of the universal primer (UP), followed by universal amplification using UPs and a regular cycle amplification.

Rapid decline of neutralizing antibodies is associated with decay of IgM in adults recovered from mild COVID-19.

The fate of protective immunity following mild severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection remains ill defined. Here, we characterize antibody responses in a cohort of participants recovered from mild SARS-CoV-2 infection with follow-up to 6 months. We measure immunoglobulin A (IgA), IgM, and IgG binding and avidity to viral antigens and assess neutralizing antibody responses over time.

Plasma From Recovered COVID-19 Patients Inhibits Spike Protein Binding to ACE2 in a Microsphere-Based Inhibition Assay.

We present a microsphere-based flow cytometry assay that quantifies the ability of plasma to inhibit the binding of spike protein to angiotensin-converting enzyme 2. Plasma from 22 patients who had recovered from mild coronavirus disease 2019 (COVID-19) and expressed anti-spike protein trimer immunoglobulin G inhibited angiotensin-converting enzyme 2-spike protein binding to a greater degree than controls. The degree of inhibition was correlated with anti-spike protein immunoglobulin G levels, neutralizing titers in a pseudotyped lentiviral assay, and the presence of fever during illness.

Plasma from recovered COVID19 subjects inhibits spike protein binding to ACE2 in a microsphere-based inhibition assay.

High throughput serological tests that can establish the presence and functional activity of anti-SARS-COV2 antibodies are urgently needed. Here we present microsphere-based Flow Cytometry assays that quantify both anti-spike IgGs in plasma, and the ability of plasma to inhibit the binding of spike protein to angiotensin converting enzyme 2 (ACE2). First, we detected anti-trimer IgGs in 22/24 and anti-RBD IgGs in 21/24 COVID+ subjects at a median of 36 (range 14-73) days following documented SARS-CoV-2 RNA (+) secretions.

Sustained Intracellular Raltegravir Depots Generated with Prodrugs Designed for Nanoparticle Delivery.

Polymeric nanocarriers have been extensively used to improve the delivery of hydrophobic drugs, but often provide low encapsulation efficiency and percent loading for hydrophilic compounds. In particular, insufficient loading of hydrophilic antiretroviral drugs such as the integrase inhibitor raltegravir (RAL) has limited the development of sustained-release therapeutics or prevention strategies against HIV. To address this, we developed a generalizable prodrug strategy using RAL as a model where loading, release and subsequent hydrolysis can be tuned by promoiety selection.

Hybrid nanocarriers incorporating mechanistically distinct drugs for lymphatic CD4 T cell activation and HIV-1 latency reversal.

A proposed strategy to cure HIV uses latency-reversing agents (LRAs) to reactivate latent proviruses for purging HIV reservoirs. A variety of LRAs have been identified, but none has yet proven effective in reducing the reservoir size in vivo. Nanocarriers could address some major challenges by improving drug solubility and safety, providing sustained drug release, and simultaneously delivering multiple drugs to target tissues and cells.

Simultaneous detection of five pig viruses associated with enteric disease in pigs using EvaGreen real-time PCR combined with melting curve analysis.

In recent years, a series of porcine diarrhea viruses such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotaviruses of group A (RVA), rotaviruses of group C (RVC), and porcine circovirus 2 (PCV2) caused enormous economic losses all over the world. While any of these viruses is capable to cause disease alone, there is often concurrent infection with more than one virus on pig farms. In this study, a multiplex real-time PCR method based on EvaGreen fluorescent dye and melting curve analysis was established to simultaneously detect these five viruses in a single closed tube.

Detection and differentiation of five diarrhea related pig viruses utilizing a multiplex PCR assay.

Porcine viral diarrhea, mainly caused by porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine group A rotaviruses (RVA), porcine group C rotaviruses (RVC) and porcine circovirus 2 (PCV2), is a serious global problem, resulting in substantial economic losses to the swine industry. For fast and reliable diagnosis of the causative agent associated with viral diarrhea in pigs, an inexpensive and easy to perform gel-based multiplex PCR assay was developed in this study to detect and differentiate the different viruses by amplicon size. The assay was able to distinguish between all five viral agents without cross-reacting with other non-target pig viruses.

Core-shell nanoparticles for targeted and combination antiretroviral activity in gut-homing T cells.

A major sanctuary site for HIV infection is the gut-associated lymphoid tissue (GALT). The α4β7 integrin gut homing receptor is a promising therapeutic target for the virus reservoir because it leads to migration of infected cells to the GALT and facilitates HIV infection. Here, we developed a core-shell nanoparticle incorporating the α4β7 monoclonal antibody (mAb) as a dual-functional ligand for selectively targeting a protease inhibitor (PI) to gut-homing T cells in the GALT while simultaneously blocking HIV infection.

Optimization and comparison of CD4-targeting lipid-polymer hybrid nanoparticles using different binding ligands.

Monoclonal antibodies and peptides are conjugated to the surface of nanocarriers (NCs) for targeting purposes in numerous applications. However, targeting efficacy may vary with their specificity, affinity, or avidity when linked to NCs. The physicochemical properties of NCs may also affect targeting.

Application of electrospun fibers for female reproductive health.

Here, we present the current challenges in women's reproductive health and the current state-of-the-art treatment and prevention options for STI prevention, contraception, and treatment of infections. We discuss how the versatile platform of electrospun fibers can be applied to each challenge, and postulate at how these technologies could be improved. The void of approved electrospun fiber-based products yields the potential to apply this useful technology to a number of medical applications, many of which are relevant to women's reproductive health.

Rational design of charged peptides that self-assemble into robust nanofibers as immune-functional scaffolds.

Unlabelled: Self-assembling peptides programed by sequence design to form predefined nanostructures are useful for a variety of biomedical applications. However, assemblies of classic ionic self-complementary peptides are unstable in neutral pH, while charged peptide hydrogels have low mechanical strength. Here, we report on the rational design of a self-assembling peptide system with optimized charge distribution and density for bioscaffold development.

In vitro-ex vivo correlations between a cell-laden hydrogel and mucosal tissue for screening composite delivery systems.

Composite delivery systems where drugs are electrospun in different layers and vary the drug stacking-order are posited to affect bioavailability. We evaluated how the formulation characteristics of both burst- and sustained-release electrospun fibers containing three physicochemically diverse drugs: dapivirine (DPV), maraviroc (MVC) and tenofovir (TFV) affect in vitro and ex vivo release. We developed a poly(hydroxyethyl methacrylate) (pHEMA) hydrogel release platform for the rapid, inexpensive in vitro evaluation of burst- and sustained-release topical or dermal drug delivery systems with varying microarchitecture.

Development and validation of a multiplex conventional PCR assay for simultaneous detection and grouping of porcine bocaviruses.

Porcine bocavirus (PBoV), a newly described porcine parvovirus, has received attention because it can be commonly identified in clinically affected pigs including pigs with post-weaning multisystemic wasting syndrome (PWMS) and pigs with diarrhea. In recent years, novel PBoVs have been identified and were classified into three genogroups, but the ability to detect and classify these novel PBoVs is not comprehensive to date. In this study, a multiplex conventional PCR assay for simultaneous detection and grouping of PBoVs was developed by screening combinations of mixed primer pairs followed by optimization of the PCR conditions.

Simultaneous measurement of etravirine, maraviroc and raltegravir in pigtail macaque plasma, vaginal secretions and vaginal tissue using a LC-MS/MS assay.

Etravirine (ETR), maraviroc (MVC) and raltegravir (RAL) are promising antiretroviral drugs being used in HIV treatment and may be interesting for prevention applications such as oral or topical pre-exposure prophylaxis. Here we describe a sensitive and accurate method for the simultaneous detection of ETR, MVC and RAL from pigtail macaque plasma, vaginal secretions, and vaginal tissue. This method is characterized by a straightforward precipitation extraction method, a limit of quantification <0.

Rapid detection and grouping of porcine bocaviruses by an EvaGreen(®) based multiplex real-time PCR assay using melting curve analysis.

Several novel porcine bocaviruses (PBoVs) have been identified in pigs in recent years and association of these viruses with respiratory signs or diarrhea has been suggested. In this study, an EvaGreen(®)-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed for simultaneous detection and grouping of novel PBoVs into the same genogroups G1, G2 and G3. Each target produced a specific amplicon with a melting peak of 81.

Coaxially electrospun fiber-based microbicides facilitate broadly tunable release of maraviroc.

Electrospun fibers show potential as a topical delivery system for vaginal microbicides. Previous reports have demonstrated delivery of anti-HIV and anti-STI (sexually transmitted infection) agents from fibers formulated using hydrophilic, hydrophobic, or pH-responsive polymers that result in rapid, prolonged, or stimuli-responsive release, respectively. However, coaxial electrospun fibers have yet to be evaluated as a highly tunable microbicide delivery vehicle.