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The COVID-19 pandemic spurred the development of innovative solutions for specimen collection and molecular detection for large-scale community testing. Among these developments is the RHINOstic nasal swab, a plastic anterior nares swab built into the cap of a standard matrix tube that facilitates automated processing of up to 96 specimens at a time. In a study of unsupervised self-collection utilizing these swabs, we demonstrate comparable analytic performance and shipping stability compared to traditional anterior nares swabs, as well as significant improvements in laboratory processing efficiency. The use of these swabs may allow laboratories to accommodate large numbers of sample collections during periods of high testing demand. Automation-friendly nasal swabs are an important tool for high-throughput processing of samples that may be adopted in response to future respiratory viral pandemics., Competing Interests: The authors have no relationship with the Rhinostics corporation. Dr. Chu reported consulting with Ellume, Pfizer, and the Bill and Melinda Gates Foundation. She has served on advisory boards for Vir, Merck, and Abbvie. She has conducted CME teaching with Medscape, Vindico, Catalyst CME, and Clinical Care Options. She has received support and reagents from Ellume and Cepheid outside of the submitted work. Dr. Englund reports consulting with AstraZeneca, Meissa Vaccines, Moderna, Pfizer, and Sanofi Pasteur, and research support from AstraZeneca, GlaxoSmithKline, Merck, Moderna, and Pfizer.

Fowl adenovirus (FAdV)-induced diseases hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH) have been affecting the poultry industry with increasing severity in the last two decades. Recently, a subunit vaccine based on a chimeric fiber protein with epitopes from different fowl adenovirus serotypes (named crecFib-4/11) has been shown to confer simultaneous protection against both HHS and IBH. However, the underlying immune mechanisms in chickens are still enigmatic, especially because of frequently absent neutralizing response despite high levels of protection. In this study, we investigated the kinetics of the humoral and cellular immune responses in specific pathogen-free chickens after vaccination with crecFib-4/11 and/or challenge with a HHS-causing strain, on a systemic level, as well as locally in target and lymphoid organs. The humoral response was assessed via enzyme-linked immunosorbent assay (ELISA) and virus neutralization test in serum, while the cellular immune response was determined by phenotyping using flow cytometry. Although vaccination induced serum antibodies, as confirmed by ELISA, such antibodies exhibited no pre-challenge neutralizing activity against FAdV-4. Nevertheless, immunized birds experienced a significant B cell increase in the liver upon challenge, remaining high throughout the experiment. Furthermore, vaccination stimulated the proliferation of cytotoxic T lymphocytes, with earlier circulation in the blood compared to the challenge control and subsequent increase in liver and spleen. Overall, these findings imply that protection of chickens from HHS after crecFib-4/11 vaccination relies on a prominent local immune response in the target organs, instead of circulating neutralizing antibodies., Competing Interests: MH and AS declare the following financial interest that may be considered as potential competing interests: Patent ‘‘Fowl adenovirus subunit vaccine and production method thereof” pending to European Patent Office. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from the Christian Doppler Research Association, in cooperation with Vaxxinova GmbH, Münster, Germany (grant nr.: 189). Vaxxinova GmbH had the following involvement in the study: preparation of vaccine formulation and mock solution (adjuvant only)., (Copyright © 2022 De Luca, Schachner, Heidl, Hess, Liebhart and Mitra.)

In the present study, we report the occurrence of several outbreaks of hepatitis in flocks of young pheasants in France, between 2017 and 2021. The disease was characterized by prostration, apathy and a median cumulative mortality of 12%, with the birds presenting multifocal to coalescing necrotizing hepatitis on necropsy. Severe extensive areas of degeneration and necrosis were observed in the liver, with degenerative hepatocytes presenting large amphophilic to acidophilic intranuclear inclusion bodies. Transmission electron microscopy examination of liver samples showed the presence of parvovirus-like virions of 21-24 nm, a finding already reported decades ago. Further investigations by Next Generation Sequencing and PCR revealed the complete genome of a novel species of parvovirus, here designated Phasianus chaphamaparvovirus 1 (PhChPV-1), that belongs to the new genus Chaphamaparvovirus in the Hamaparvovirinae subfamily. In situ hybridization and real-time PCR confirmed the etiology of the outbreaks, demonstrating the viral genome in the lesions. The findings establish the etiology of a pathology first described in pheasants 50 years ago and pave the way for a targeted protection strategy., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

In the past decades, fowl adenovirus (FAdV)-related diseases became an increasing concern for the poultry industry worldwide. Various immunization strategies against FAdVs have been experimentally investigated, with a particular focus on subunit vaccines against hepatitis-hydropericardium syndrome (HHS), caused by FAdV serotype 4, and inclusion body hepatitis (IBH), caused by serotypes 2, 8a, 8b and 11. In this study, we extended our innovative concept of recombinant chimeric fiber proteins to design a novel chimera combining epitopes from two distinct serotypes, FAdV-4 and -11, and we investigated its efficacy to simultaneously protect chickens against HHS and IBH. Specific pathogen-free chickens were vaccinated with the novel recombinant chimeric fiber and subsequently challenged with either a HHS- or IBH-causing strain. Vaccinated/challenged birds exhibited a reduction of clinical signs, limited hepatomegaly and lower levels of AST compared to the respective challenge controls. Furthermore, the vaccine prevented atrophy of HHS-affected lymphoid organs, such as thymus and bursa of Fabricius, and viral load in the target organs was significantly reduced. Clinical protection was associated with high levels of pre-challenge antibodies measured on ELISA plates coated with the vaccination antigen. Interestingly, the development of neutralizing antibodies was limited against FAdV-11 and absent against FAdV-4, indicating that protection granted by such an antigen may be linked to different immunization pathways. In conclusion, we proved that the concept of chimeric fiber vaccines can be extended across viral species boundaries and represents the first single-component FAdV subunit vaccine providing comprehensive protection against different FAdV-associated diseases., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michael Hess and Anna Schachner have patent “Fowl adenovirus subunit vaccine and production method thereof” pending to European Patent Office., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Vaccines against inclusion body hepatitis in chickens are complicated by the involvement of antigenically diverse fowl adenovirus types. Though immunization with fiber protein confers robust protection, type specificity of fiber antibodies is an obstacle for the desired broad coverage. In this study, we utilized information on multiple linear epitopes predicted in the Fowl Aviadenovirus E (FAdV-E) fiber head (knob) to develop chimeric fibers with an exchange between two serotypes' sequences, each containing proposed epitopes. Two consecutive segments pertaining to amino acid positions 1 to 441 and 442 to 525/523 in the fibers of FAdV-8a and -8b, types of Fowl Aviadenovirus E that cause inclusion body hepatitis, were swapped reciprocally to result in novel chimeras, crecFib-8a/8b and crecFib-8b/8a. crecFib was indistinguishable from monospecific recombinant fibers in its eactivity with different FAdV antisera in Western blotting. However, contrary to the results for monospecific fibers, crecFib induced cross-neutralizing antibodies against both serotypes in chickens. This demonstrates three nonidentical epitopes in the FAdV-E fiber, the conserved epitope detected in Western blotting and at least two epitopes participating in neutralization, being type specific and located opposite residue position 441-442. Furthermore, we supply conformational evidence for a site in the fiber knob with accessibility critical for neutralization. With such an extended neutralization spectrum compared to those of individual fibers, crecFib was anticipated to fulfill and even extend the mechanistic basis of fiber-mediated protection toward bivalent coverage. Accordingly, crecFib, administered as a single-antigen component, protected chickens simultaneously against challenge with FAdV-8a or -8b, demonstrated by up-to-complete resistance to clinical disease, prevention of target organ-related changes, and significant reduction of viral load. IMPORTANCE The control of inclusion body hepatitis, a disease of economic importance for chicken production worldwide, is complicated by an etiology involving multiple divergent fowl adenovirus types. The fiber protein is principally efficacious in inducing neutralizing and protective antibodies in vaccinated chickens; however, it faces limitations due to its intrinsic type specificity for neutralization. In this study, based on an in silico -guided prediction of multiple epitopes in the fowl adenovirus fiber head's loops, we designed chimeric proteins, swapping N- and C-distal fiber portions, each containing putative epitopes, between divergent types FAdV-8a and -8b. In in vitro and in vivo studies, the chimeric fiber displayed extended properties compared to those of individual monotype-specific fibers, allowing the number, distribution, functionality, and conformational bearings of epitopes of the fowl adenovirus fiber to be characterized in more detail. Importantly, the chimeric fiber induced cross-neutralizing antibodies and protective responses in chickens against infections by both serotypes, promoting the advancement of broadly protective subunit vaccination strategies against FAdV.

Background: The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse-transcription PCR (RT-qPCR). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce. To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction., Methods: We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral inactivation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance., Results: After optimization, SwabExpress has a low limit of detection at 2-4 molecules/µL, 100% sensitivity, and 99.4% specificity when compared side by side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time., Conclusion: SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics., (© American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A recombinant fowl adenovirus (FAdV) fiber protein, derived from a FAdV-8a strain, was tested for its efficacy to protect chickens against inclusion body hepatitis (IBH). FAdV-E field isolates belonging to both a homotypic (FAdV-8a) and heterotypic (-8b) serotype were used as challenge. Mechanisms underlying fiber-induced protective immunity were investigated by fiber-based ELISA, virus neutralization assays and flow cytometry of peripheral blood mononuclear cells, monitoring the temporal developments of humoral and cellular responses after vaccination and challenge exposure. Birds were clinically protected from the homologous challenge and showed a significant reduction of viral load in investigated target organs, whereas fiber-based immunity failed to counteract the heterologous serotype infection. These findings were supported in vitro by the strictly type-specific neutralizing activity of fiber immune sera. In protected birds, fiber vaccination prevented a post-challenge drop of peripheral B cells in blood. Furthermore, fiber immunization stimulated CD4 + T lymphocyte proliferation while moderating the CD8α + T cell response and prevented challenge-induced changes in systemic monocytes/macrophages and γδ + T cell subpopulations. Both vaccinated and adjuvant-only injected birds experienced a priming of systemic B cells and TCRγδ + T lymphocytes, which masked possible pre-challenge effects due to the antigen. In conclusion, within FAdV-E, recombinant fiber represents a vaccine candidate to control the adverse effects of homotypic infection by eliciting an effective humoral immunity and regulating B and T cell response, whereas the failure of heterotypic protection suggests a primordial role of humoral immunity for this vaccine.

The airway epithelium is a central player in the defense against pathogens including efficient mucociliary clearance and secretion of immunoglobulins, mainly polymeric IgA, but also IgG. Pulmonary administration of therapeutic antibodies on one hand, and intranasal immunization on the other, are powerful tools to treat airway infections. In either case, the airway epithelium is the primary site of antibody transfer. In various epithelia, bi-polar transcytosis of IgG and IgG immune complexes is mediated by the human neonatal Fc receptor, FcRn, but FcRn expression in the nasal epithelium had not been demonstrated, so far. We prepared affinity-purified antibodies against FcRn α-chain and confirmed their specificity by Western blotting and immunofluorescence microscopy. These antibodies were used to study the localization of FcRn α-chain in fixed nasal tissue. We here demonstrate for the first time that ciliated epithelial cells, basal cells, gland cells, and endothelial cells in the underlying connective tissue express the receptor. A predominant basolateral steady state distribution of the receptor was observed in ciliated epithelial as well as in gland cells. Co-localization of FcRn α-chain with IgG or with early sorting endosomes (EEA1-positive) but not with late endosomes/lysosomes (LAMP-2-positive) in ciliated cells was observed. This is indicative for the presence of the receptor in the recycling/transcytotic pathway but not in compartments involved in lysosomal degradation supporting the role of FcRn in IgG transcytosis in the nasal epithelium.

The leaf beetle Chrysomela aeneicollis occurs across Western North America, either at high elevation or in small, isolated populations along the coast, and thus has a highly fragmented distribution. DNA sequence data (three loci) were collected from five regions across the species range. Population connectivity was examined using traditional ecological niche modeling, which suggested that gene flow could occur among regions now and in the past. We developed geographically explicit coalescence models of sequence evolution that incorporated a two-dimensional representation of the hypothesized ranges suggested by the niche-modeling estimates. We simulated sequence data according to these models and compared them to observed sequences to identify most probable scenarios regarding the migration history of C. aeneicollis. Our results disagreed with initial niche-modeling estimates by clearly rejecting recent connectivity among regions, and were instead most consistent with a long period of range fragmentation, extending well beyond the last glacial maximum. This application of geographically explicit models of coalescence has highlighted some limitations of the use of climatic variables for predicting the present and past range of a species and has explained aspects of the Pleistocene evolutionary history of a cold-adapted organism in Western North America., (© 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.)

Purpose: Early metastasis in node-negative breast cancer indicates that breast cancer cells obviously can bypass the lymph nodes and disseminate directly hematogenous to distant organs. For this purpose, we evaluated the prognostic value of blood-borne, HER2-positive circulating tumor cells (CTC) in the peripheral blood from 42 breast cancer patients with a median follow-up of 95 months., Experimental Design: Cells were isolated by the patented combined buoyant density gradient and immunomagnetic separation procedure and analyzed by immunocytochemistry., Results: We detected one to eight CTCs in the peripheral blood of 17 of 35 patients (48.6%) presenting no overt metastasis. As a positive control, 7 of 7 (100%) patients with metastatic disease presented positive. Healthy persons and patients (n = 32) operated for nonmalignant diseases presented negative for CTCs. The presence and frequency of HER2-positive CTCs correlated with a significantly decreased disease-free survival (P < 0.005) and overall survival (P < 0.05). Interestingly, in 12 patients with HER2-positive CTCs, the primary tumor was negative for HER2 as assessed by immunohistochemical score and fluorescence in situ hybridization., Conclusions: This study provides some evidence of a prognostic effect of HER2-positive CTCs in stage I to III breast cancer. Future studies have to determine the outcome of patients treated with HER2-targeting therapies with respect to HER2-positive CTC levels because it is not unlikely that high levels of HER2-positive CTCs reflect the activity of the tumor and may predict response to trastuzumab.

Clinical studies including thousands of breast cancer patients have shown that c-erbB-2 is amplified and overexpressed in 20-30% of invasive human breast cancers and that it is associated with distant metastasis in specified patient subgroups. To isolate and characterize hematogeneously spreading c-erbB-2-positive epithelium-derived cells from the peripheral blood of breast cancer patients, a combined buoyant density gradient and immuno-magnetic separation method has been used. The method utilizes a biotinylated anti-cytokeratin monoclonal antibody (MAb) for capturing the epithelium-derived cells. The expression of c-erbB-2 by the captured cells was detected using an anti-c-erbB-2 rabbit antibody (21N) coupled to an anti-rabbit gold-labeled anti-body, whereby immunoenzymatic cytokeratin staining was performed using a silver-enhanced immunogold double staining protocol. In total, 29 of the 46 patients tested had either cytokeratin (24/29) or cytokeratin/c-erbB-2 (19/29) positive clustered cells in their peripheral blood. We thus report here the presence and the frequency of clone-specifically stained clustered cells in the peripheral blood of breast cancer patients. The frequency of cytokeratin/c-erbB2 double-positive clustered cells in the peripheral blood was on average 10 times higher than that of double-positive single cells. The numbers of cytokeratin/c-erbB-2 double-positive clustered cells were positively correlated with the stage of tumors. Results of in vitro motility experiments using single and clustered cells from primary breast cancer tissue strongly support the assumption that cytokeratin/c-erbB-2 double-positive clustered cells have a high potential for locomotion. We suggest that blood-borne epithelium-derived c-erbB-2-positive clustered cells are the possible precursor cells responsible for the formation of distant metastases and bone marrow micrometastases.

The detection of blood-borne prostate cancer (PCA) cells may help with clinical staging and the further understanding of PCA metastases. We discovered prostate-specific antigen (PSA)-positive stained but not PSA mRNA-expressing blood cells by means of cell sorting and PSA reverse transcription-PCR in patients. Therefore, we developed a cytokeratin immunomagnetic method to isolate PSA-positive epithelial cells from the circulating blood of PCA patients. We obtained blood-borne single cells from 6 of 10 PCA patients and clustered cells from 8 of 10 PCA patients. Patients with benign prostate hyperplasia tested negative for cell clusters. The reported isolation method yielded prostate-derived cells or clusters of them from PCA-diagnosed patients.