Identification of memory mechanism in tissue-resident stem cells via ANGPTL4 beyond immune cells upon viral antigen exposure.

Although memory functions of immune cells characterized by increased resistance to subsequent infections after initial pathogen exposure are well-established, it remains unclear whether non-immune cells, especially tissue-resident stem cells, exhibit similar memory mechanisms. The present study revealed that detrimental effects of initial viral antigen exposure (human papillomavirus [HPV]) on diverse stem cell functions were significantly exacerbated upon subsequent secondary exposure both in vitro and in vivo. Importantly, endometrial stem cells exhibited robust memory functions following consecutive HPV antigen exposures, whereas fully differentiated cells such as fibroblasts and vesicular cells did not show corresponding changes in response to the same antigen exposures. Deficiency of angiopoietin-like 4 (ANGPTL4) achieved through small hairpin RNA knockdown in vitro and knockout (KO) mice in vivo highlighted the critical role of ANGPTL4 in governing memory functions associated with various stem cell processes. This regulation occurred through histone H3 methylation alterations and PI3K/Akt signaling pathways in response to successive HPV antigen exposures. Furthermore, memory functions associated with various stem cell functions that were evident in wild-type mice following consecutive exposures to HPV antigen were not observed in ANGPTL4 KO mice. In summary, our findings strongly support the presence of memory mechanism in non-immune cells, particularly tissue-resident stem cells.

Microneme-located VP2 in Eimeria acervulina elicits effective protective immunity against infectious bursal disease virus.

Using transgenic Eimeria spp. to deliver exogenous antigens is a viable option for developing multivalent live vaccines. Previous research revealed that the location of antigen expression in recombinant Eimeria dictates the magnitude and type of immune responses. In this study, we constructed genetically modified Eimeria acervulina that expressed VP2 protein, a protective antigen from infectious bursal disease virus (IBDV), on the surface or in the microneme of sporozoites. After vaccination, VP2-specific antibody was readily detected in specific pathogen-free chickens receiving transgenic E. acervulina parasites expressing VP2 in microneme, but animals vaccinated with which expressing VP2 on surface failed to produce detectable antibody after two times immunizations. Moreover, the bursal lesion of microneme-located VP2 transgenic E. acervulina immunized chickens was less severe compared with un-immunized animals after IBDV challenge infection. Therefore, genetically modified E. acervulina that express IBDV-derived VP2 in micronemes are effective in inducing specific antibody responses against VP2, while parasites that have VP2 expression on cell surface are not suitable. Thus, the use of Eimeria parasites as vaccine vectors needs to consider the proper targeting of exogenous immunogens. Our results have implications for the design of other vector vaccines.

Postacute COVID-19 is Characterized by Gut Viral Antigen Persistence in Inflammatory Bowel Diseases.

BACKGROUND & AIMS: The coronavirus disease 2019 (COVID-19) pandemic has affected populations, societies, and lives for more than 2 years. Long-term sequelae of COVID-19, collectively termed the postacute COVID-19 syndrome, are rapidly emerging across the globe. Here, we investigated whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen persistence underlies the postacute COVID-19 syndrome. METHODS: We performed an endoscopy study with 46 patients with inflammatory bowel disease (IBD) 219 days (range, 94-257) after a confirmed COVID-19 infection. SARS-CoV-2 antigen persistence was assessed in the small and large intestine using quantitative polymerase chain reaction of 4 viral transcripts, immunofluorescence of viral nucleocapsid, and virus cultivation from biopsy tissue. Postacute COVID-19 was assessed using a standardized questionnaire, and a systemic SARS-CoV-2 immune response was evaluated using flow cytometry and enzyme-linked immunosorbent assay at endoscopy. IBD activity was evaluated using clinical, biochemical, and endoscopic means. RESULTS: We report expression of SARS-CoV-2 RNA in the gut mucosa ∼7 months after mild acute COVID-19 in 32 of 46 patients with IBD. Viral nucleocapsid protein persisted in 24 of 46 patients in gut epithelium and CD8+ T cells. Expression of SARS-CoV-2 antigens was not detectable in stool and viral antigen persistence was unrelated to severity of acute COVID-19, immunosuppressive therapy, and gut inflammation. We were unable to culture SARS-CoV-2 from gut tissue of patients with viral antigen persistence. Postacute sequelae of COVID-19 were reported from the majority of patients with viral antigen persistence, but not from patients without viral antigen persistence. CONCLUSION: Our results indicate that SARS-CoV-2 antigen persistence in infected tissues serves as a basis for postacute COVID-19. The concept that viral antigen persistence instigates immune perturbation and postacute COVID-19 requires validation in controlled clinical trials.

Contribution of High Viral Loads, Detection of Viral Antigen and Seroconversion to Severe Acute Respiratory Syndrome Coronavirus 2 Infectivity.

BACKGROUND: From a public health perspective, effective containment strategies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) should be balanced with individual liberties. METHODS: We collected 79 respiratory samples from 59 patients monitored in an outpatient center or in the intensive care unit of the University Hospital Regensburg. We analyzed viral load by quantitative real-time polymerase chain reaction, viral antigen by point-of-care assay, time since onset of symptoms, and the presence of SARS-CoV-2 immunoglobulin G (IgG) antibodies in the context of virus isolation from respiratory specimens. RESULTS: The odds ratio for virus isolation increased 1.9-fold for each log10 level of SARS-CoV-2 RNA and 7.4-fold with detection of viral antigen, while it decreased 6.3-fold beyond 10 days of symptoms and 20.0-fold with the presence of SARS-CoV-2 antibodies. The latter was confirmed for B.1.1.7 strains. The positive predictive value for virus isolation was 60.0% for viral loads >107 RNA copies/mL and 50.0% for the presence of viral antigen. Symptom onset before 10 days and seroconversion predicted lack of infectivity with negative predictive values of 93.8% and 96.0%. CONCLUSIONS: Our data support quarantining patients with high viral load and detection of viral antigen and lifting restrictive measures with increasing time to symptom onset and seroconversion. Delay of antibody formation may prolong infectivity.

A preliminary study of the localization of infectious laryngotracheitis virus glycoprotein E within specific peripheral blood lymphocytes.

Infectious laryngotracheitis virus (ILTV) DNA has been detected in blood fractions, but the cell phenotype with which the virus is associated is unknown. This study investigated the presence of ILTV antigen in peripheral blood cells of six acutely ILTV-infected chickens (5 or 9 days post ocular inoculation with a virulent isolate) and three sham-inoculated chickens using immunofluorescent staining. Blood fractions were separated by Ficoll-Paque density gradient centrifugation, and smears were prepared from erythrocyte and leukocyte fractions. The smears were stained for ILTV glycoprotein E and the leukocyte markers CD4, CD8, Bu-1 (B cell), KUL01 (monocyte/macrophage), TCRγδ, and TCRαß/Vß2 and examined under a confocal microscope. In samples from infected birds, ILTV gE-specific fluorescence was localized in B cells and all evaluated T cell types, but not in monocytes and erythrocytes. The percentage of CD4, CD8, TCRγδ, TCRαß/Vß1, TCRαß/Vß2 and B cells positive for ILTV antigen ranged from 13.3% to 22.3%. None of the samples from the sham-inoculated chickens exhibited fluorescence for ILTV gE. The results of this pilot study suggest that ILTV has a tropism for peripheral blood T and B cells. Further research is required to investigate whether these cells support ILTV productive replication. RESEARCH HIGHLIGHTSSelective tropism of ILTV for peripheral blood cells was demonstrated in acutely infected birds.The ILTV antigen gE was detected in blood CD4, CD8, TCRγδ, TCRαß and B cells but not in monocytes and erythrocytes.The highest percentage of ILTV antigen was observed in CD4 cells (22.3%) followed by TCRαß/Vß1 (20.6%), CD8 (15.4%), TCRαß/Vß2 or B cells (14.4%) and TCRγδ cells (13.3%).

Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Tracking.

BACKGROUND: High-sensitivity severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations. METHODS: We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection of 0.5 pg/mL (10.6 fmol/L) nucleocapsid antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives and quantified and tracked antigen dynamics in intensive care unit coronavirus disease 2019 (COVID-19) inpatients and immunocompromised COVID-19 patients. RESULTS: Compared to qualitative reverse transcription-polymerase chain reaction methods, the MSAA demonstrated a positive percentage agreement of 97% (95% CI 92%-99%) and a negative percentage agreement of 97% (95% CI 94%-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with cycle threshold values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden. CONCLUSIONS: The MSAA enables sensitive and specific detection of acute infections and quantification and tracking of antigen burden and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.

A systematic review of the sensitivity and specificity of lateral flow devices in the detection of SARS-CoV-2.

BACKGROUND: Lateral flow devices (LFDs) are viral antigen tests for the detection of SARS-CoV-2 that produce a rapid result, are inexpensive and easy to operate. They have been advocated for use by the World Health Organisation to help control outbreaks and break the chain of transmission of COVID-19 infections. There are now several studies assessing their accuracy but as yet no systematic review. Our aims were to assess the sensitivity and specificity of LFDs in a systematic review and summarise the sensitivity and specificity of these tests. METHODS: A targeted search of Pubmed and Medxriv, using PRISMA principles, was conducted identifying clinical studies assessing the sensitivity and specificity of LFDs as their primary outcome compared to reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of SARS-CoV-2. Based on extracted data sensitivity and specificity was calculated for each study. Data was pooled based on manufacturer of LFD and split based on operator (self-swab or by trained professional) and sensitivity and specificity data were calculated. RESULTS: Twenty-four papers were identified involving over 26,000 test results. Sensitivity from individual studies ranged from 37.7% (95% CI 30.6-45.5) to 99.2% (95% CI 95.5-99.9) and specificity from 92.4% (95% CI 87.5-95.5) to 100.0% (95% CI 99.7-100.0). Operation of the test by a trained professional or by the test subject with self-swabbing produced comparable results. CONCLUSIONS: This systematic review identified that the performance of lateral flow devices is heterogeneous and dependent on the manufacturer. Some perform with high specificity but a great range of sensitivities were shown (38.32-99.19%). Test performance does not appear dependent on the operator. Potentially, LFDs could support the scaling up of mass testing to aid track and trace methodology and break the chain of transmission of COVID-19 with the additional benefit of providing individuals with the results in a much shorter time frame.

Ultra-Sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression. METHODS: We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of patients with coronavirus disease (COVID-19). We studied plasma from 64 patients who were COVID-19 positive, 17 who were COVID-19 negative, and 34 prepandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 patients with COVID-19. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes. RESULTS: SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95% CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral-antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within 1 day) was statistically significant. CONCLUSIONS: The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of patients who were COVID-19 positive to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease.

Chronic hepatitis B: The interplay between intrahepatic lymphocyte population and viral antigens in relation to liver damage.

In Chronic hepatitis B (CHB) infection, virus and immune response interplay is thought to be responsible for pathogenesis. Yet, the impact of each immune cell population and viral protein expression in liver damage is still unknown. Our aim was to study the interplay between intrahepatic immune response and viral activity in relation to CHB liver damage. Immunostaining was performed in 29 liver biopsies from untreated CHB patients to characterize liver infiltrate [Th (CD4+), CTL (CD8+), Treg (FoxP3+), Th17 (IL-17A+) and Th1 (T-bet+)] and viral antigen expression (HBsAg and HBcAg). Inflammatory activity and fibrosis were assessed using the HAI and METAVIR scoring system. All studied populations were identified in the portal-periportal (P-P) areas with a CD4+ lymphocyte predominance, while only CD8+ and FoxP3+ cells were observed in the intralobular area. Both P-P CD4+ and intralobular CD8+ cell frequencies were increased among severe hepatitis cases. Concerning HBsAg and HBcAg expression, a mutually exclusive pattern was observed. HBcAg was mainly detected among HBeAg-positive patients and was associated with hepatitis severity and higher frequency of P-P FoxP3+, intralobular CD8+ and FoxP3+ cells. HBsAg was identified among HBeAg-negative cases with less severe hepatitis grade and lower frequency of P-P CD4+ and intralobular FoxP3+ lymphocytes. In conclusion, the HBV antigen profile expression seen during CHB infection may be reflecting different stages of viral replication which impacts the host immune response and liver damage process. While HBcAg might be an inducer of a regulatory microenvironment, the intralobular CTL population seemed to have a key role in hepatitis severity.

Diagnostic tests for human rabies. / Tests diagnostiques de la rage chez l'homme.

The lack of reliable data concerning the number of human deaths from rabies presents one of the principal difficulties in a realistic assessment of the importance of this disease, and this lack of an accurate assessment has led to its underestimation and neglect. Priority should therefore be given to establishing a diagnostic test that can confirm human rabies on the basis of biological results. Indeed, only a laboratory diagnosis can properly identify infection, because clinical diagnosis remains difficult to interpret and is insufficiently specific. Historically, diagnosis has been based solely on post-mortem analysis of a cerebral biopsy using immunofluorescence techniques. Although this remains the standard method, considerable progress has been made with the advent of new molecular techniques and the evaluation of new, less-invasive sampling methods that are more easily accepted by the patient's family. Intra-vitam diagnosis of human rabies is now possible using reliable, robust, validated techniques that can be used everywhere, including in regions with limited resources, using minimally invasive or non-invasive sampling (such as saliva or skin biopsies). In practice, one of the major challenges with the diagnosis of human rabies is still the transfer and accessibility of such validated techniques in centralised reference laboratories located in low-income enzootic countries, in order to achieve the biological confirmation of each suspected case of rabies. At the same time, it is necessary to develop easy, fast and low-cost diagnostic methods that can be used in rural and remote areas in peripheral laboratories, or ideally at the patient's bedside.

L'absence de données fiables concernant le nombre de décès humains dus à la rage représente l'une des limitations majeures à l'évaluation réelle du poids mondial de cette maladie, contribuant ainsi à sa sous-estimation et à son caractère négligé. Devant ce constat, l'établissement d'un diagnostic de confirmation de la rage chez l'homme basé sur des résultats biologiques doit être favorisé. En effet, seul le diagnostic de laboratoire permet de valider l'infection, le diagnostic clinique restant difficile d'interprétation et insuffisamment spécifique. Historiquement, ce diagnostic était réalisé exclusivement au stade post-mortem via l'analyse d'une biopsie cérébrale par technique d'immunofluorescence. Bien qu'il s'agisse encore de la méthode de référence, des progrès considérables ont été faits, avec l'avènement de nouvelles techniques moléculaires et l'évaluation de nouveaux types de prélèvements moins invasifs et facilement acceptés par les proches du patient. Ces progrès autorisent maintenant la mise en oeuvre d'un diagnostic intra-vitam de la rage chez l'homme basé sur des techniques fiables, robustes et validées et pouvant être utilisées à tout niveau y compris dans les zones à ressources limitées à partir de prélèvements peu ou non invasifs (tels la salive ou les biopsies de peau). En effet, l'un des enjeux majeurs du diagnostic de la rage chez l'homme réside aussi dans le transfert et l'accessibilité de ces techniques validées, au niveau des laboratoires de référence situés dans les pays enzootiques à faible revenu, afin de réaliser une confirmation biologique de chaque cas suspect de rage. En parallèle, il est nécessaire de poursuivre les recherches sur le développement de méthodes de diagnostic simplifiées, rapides et de faible coût pouvant être utilisées de façon délocalisée, dans les laboratoires périphériques en zone rurale, voire au lit du patient.

La ausencia de datos fidedignos sobre el número de personas fallecidas a causa de la rabia constituye una de las principales limitaciones a la hora de evaluar con exactitud la carga mundial que impone la enfermedad, lo que contribuye al hecho de que esté subestimada y, por consiguiente, desatendida. De semejante constatación se desprende la necesidad de favorecer la instauración de un diagnóstico de confirmación de la rabia humana basado en resultados biológicos, en la medida en que el diagnóstico de laboratorio es el único modo de validar la presencia de la infección, pues el diagnóstico clínico presenta dificultades de interpretación y no es lo bastante específico. Históricamente este diagnóstico se realizaba únicamente tras la muerte del individuo, mediante el análisis por inmunofluorescencia de una muestra encefálica. Aunque este sigue siendo el método de referencia, el advenimiento de nuevas técnicas moleculares y el estudio de nuevos tipos de muestras, obtenidas por métodos menos invasivos y fácilmente aceptados por los allegados del paciente, han deparado progresos considerables, que permiten hoy realizar un diagnóstico intra-vitam de la rabia humana utilizando técnicas fiables, robustas y validadas que se pueden aplicar en todos los niveles, incluso en zonas con escasos recursos, a partir de muestras obtenidas por procedimientos poco o nada invasivos (muestras de saliva o biopsias de piel). Uno de los principales envites del diagnóstico de la rabia en el ser humano reside, en efecto, en la accesibilidad y la transferencia de estas técnicas validadas a laboratorios de referencia situados en los países enzoóticos de renta baja para poder realizar en ellos una confirmación biológica de todo caso sospechoso de rabia. Paralelamente, es necesario seguir investigando para instituir métodos de diagnóstico simplificados, rápidos y poco costosos que se puedan aplicar de forma descentralizada, esto es, en los laboratorios periféricos de zonas rurales e incluso junto al lecho del paciente.

Viral exanthems: An update on laboratory testing of the adult patient.

Although classic viral exanthems of childhood are well described, they are rarely differentiated in adults. Laboratory techniques for viral identification have advanced without substantial literature to suggest how a dermatologist ought to conduct a cost-effective and diagnostic viral panel. Certain clinical features such as petechiae, vesicles, and dusky macular or morbilliform exanthems point strongly toward a viral exanthem. Differentiation of drug and viral causes of morbilliform eruptions has proven difficult. It is possible that with further diagnostic refinement that unnecessary and fruitless workups of an exanthem and unneeded discontinuation of drugs can be avoided. We review viral exanthems based on clinical features and discuss the available and optimal laboratory techniques to assist the dermatologist in a targeted workup.

HCV core and NS3 proteins mediate toll like receptor induced innate immune response in corneal epithelium.

Direct association of dry eye syndrome and hepatitis C virus (HCV) infection is a well established fact. In this context, the current study examines the in vitro corneal inflammatory response with respect to HCV core and NS3 antigens. Toll like receptors (TLRs) are pattern recognition receptors which can mediate innate immune response. In the present study, corneal epithelial cells responded to HCV core and NS3 proteins by secreting pro-inflammatory cytokines IL-8, IL-6 and TNF-α via TLR1, TLR2 and TLR6 mediated innate immune response. MyD88/NF-kB signalling was involved in pro-inflammatory cytokine production. Corneal epithelium synthesised nitric oxide (NO) via iNOS during HCV core and NS3 exposure. On later stages of inflammation, cells underwent apoptosis which lead to cell death. SiRNA mediated silencing of TLR1, TLR2 and TLR6 resulted in a significant down regulation of IL-8 and NO. In conclusion, this study indicates that HCV core and NS3 proteins are capable of inducing immune response in corneal epithelium which can potentiate the pathology of HCV associated dry eye condition. Blocking specific TLR response can have therapeutic application in controlling the inflammatory response associated with this dry eye condition.

Oral delivery of Eimeria acervulina transfected sequentially with two copies of the VP2 gene induces immunity against infectious bursal disease virus in chickens.

Chicken coccidiosis caused by Eimeria spp. can occur on almost all poultry farms, causing huge economic losses to the industry. Genetically manipulated Eimeria parasites as a vaccine vector to deliver viral antigens have been reported. In our preliminary study, transgenic E. acervulina expressing a VP2 gene (Ea-VP2) of the infectious bursal disease virus (IBDV) demonstrated partial protection against IBDV infection. To enhance immune responses, we aimed to increase the VP2 gene copy number in transgenic E. acervulina. In this study, we used a novel plasmid vector carrying a VP2 gene fused with three flag tags and a red fluorescent reporter gene (mCherry). The vector was introduced into Ea-VP2 sporozoites through nucleofection, leading to the generation of Ea-2VP2. Subsequent analysis revealed a notable escalation in the fluorescent rate, increasing from 0.11 to 95.1% following four consecutive passages facilitated by fluorescent-activated cell sorting. Verification via PCR, Western blot, and immunofluorescence confirmed the successful construction of the Ea-2VP2 population. Despite lower fecundity compared to wild-type E. acervulina, Ea-2VP2 maintained immunogenicity. Our research effectively created a transgenic E. acervulina strain transfected sequentially with two copies of the VP2 gene from IBDV. This modification resulted in an increased humoral immune response after primary immunization in chickens. Additionally, it demonstrated a degree of protection within the bursa against IBDV infection. Future studies will focus on further enhancing immune response levels.

Leveraging oncovirus-derived antigen against the viral malignancies in adoptive cell therapies.

Adoptive cell therapies (ACTs) have revolutionized cancer immunotherapy, prompting exploration into their application against oncoviruses. Oncoviruses such as human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), and Epstein-Barr virus (EBV) contribute significantly (12-25%) to human malignancies through direct or indirect oncogenic mechanisms. These viruses persistently or latently infect cells, disrupt cellular homeostasis and pathways, challenging current antiviral treatment paradigms. Moreover, viral infections pose additional risks in the setting of long-term cancer therapy and lead to morbidity and mortality. Virally encoded oncoproteins, which are tumor-restricted, immunologically foreign, and even uniformly expressed, represent promising targets for patient-tailored ACTs. This review elucidates the rationale for leveraging viral antigen-specific ACTs in combating viral-associated malignancies. On this basis, ongoing preclinical studies consolidate our understanding of harnessing ACTs against viral malignancies, underscoring their potential to eradicate viruses implicated in cancer progression. Furthermore, we scrutinize the current landscape of clinical trials focusing on virus-specific ACTs and discuss their implications for therapeutic advancement.

The HLA-II immunopeptidome of SARS-CoV-2.

Targeted synthetic vaccines have the potential to transform our response to viral outbreaks, yet the design of these vaccines requires a comprehensive knowledge of viral immunogens. Here, we report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peptides that are naturally processed and loaded onto human leukocyte antigen-II (HLA-II) complexes in infected cells. We identify over 500 unique viral peptides from canonical proteins as well as from overlapping internal open reading frames. Most HLA-II peptides colocalize with known CD4+ T cell epitopes in coronavirus disease 2019 patients, including 2 reported immunodominant regions in the SARS-CoV-2 membrane protein. Overall, our analyses show that HLA-I and HLA-II pathways target distinct viral proteins, with the structural proteins accounting for most of the HLA-II peptidome and nonstructural and noncanonical proteins accounting for the majority of the HLA-I peptidome. These findings highlight the need for a vaccine design that incorporates multiple viral elements harboring CD4+ and CD8+ T cell epitopes to maximize vaccine effectiveness.

Production and characterization of egg yolk antibodies against the ZIKV NS2B expressed in Nicotiana benthamiana.

The emergence of Zika virus (ZIKV) and its associated neonatal and congenital complications pose a threat to global health, particularly in tropical and subtropical regions with co-circulation of related flaviviruses and intense vector proliferation. Diagnosis of ZIKV by RT-PCR is limited to the viraemic phase and is not always accessible in low-income tropical settings, while serological tests often show cross-reactivity with other flaviviruses. Given the similarity of ZIKV symptoms to those of other arboviruses, but the different prognosis and risks, it is important to develop specific and accessible diagnostic tools. Egg yolk antibodies (IgY) were obtained from Leghorn laying hens immunized with recombinant ZIKV NS2B protein produced in agroinfiltrated Nicotiana benthamiana. After three immunizations, total IgY was recovered from the eggs by the 20% ammonium sulfate precipitation method. After characterisation by SDS-PAGE, dot blotting and ELISA, the IgY was adsorbed to dengue virus (DENV) from cell culture supernatants and tested for its ability to specifically detect ZIKV-positive sera samples. High yield and purity were observed on SDS-PAGE for polyclonal IgY, which reacted with NS2B at high titres in ELISA and detected both NS2B and ZIKV in dot blotting. However, a cross-reaction with DENV was observed and the anti-NS2B IgY was unable to discriminate ZIKV from DENV positive sera samples, even after adsorption with DENV. This is probably due to the phylogenetic relationship of the viruses and the shared identity of their proteins.

Influence of seasonal and operator variations on diagnostic accuracy of lateral flow devices during the COVID-19 pandemic: a systematic review and meta-analysis.

BACKGROUND: Lateral flow tests (LFT) are point-of-care rapid antigen tests that allow isolation and control of disease outbreaks through convenient, practical testing. However, studies have shown significant variation in their diagnostic accuracy. We conducted a systematic review of the diagnostic accuracy of LFTs for the detection of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) to identify potential factors affecting their performance. METHODS: A systematic search of online databases was carried out to identify studies assessing the sensitivity and specificity of LFTs compared with polymerase chain reaction (PCR) tests. Data were extracted and used to calculate pooled sensitivity and specificity. Meta-regression analysis was conducted to identify covariates influencing diagnostic accuracy. RESULTS: In total, 76 articles with 108,820 test results were identified for analysis. Pooled sensitivity and specificity were 72% (95% confidence interval (CI): 0.68-0.76) and 100% (95% CI: 0.99-1.00), respectively. Staff operation of the LFT showed a statistically significant increase in sensitivity (p=0.04) and specificity (p=0.001) compared with self-operation by the test subjects. The use of LFTs in symptomatic patient subgroups also resulted in higher test sensitivity. CONCLUSION: LFTs display good sensitivity and extremely good specificity for SARS-CoV-2 antigen detection; they become more sensitive in patients with symptoms and when performed by trained professionals.

Viral Titer Quantification of West Nile Virus by Immunostaining Plaque Assay.

Immunostained plaque assay based on the specific antibody binding to viral antigen enables the detection and titration of virus infectivity, especially for viruses that could not form plaques using the classical crystal violet or neutral red staining methods. Here we describe the application of this method to quantify viral titers of wild-type West Nile virus (WNV-WT) and replication-defective WNV-ΔNS1 virus.

A longitudinal study to detect hepatitis B surface and core-related antigens in chronic hepatitis B patients with hepatitis B surface antigen seroclearance using highly sensitive assays.

BACKGROUND: This study aimed to evaluate the usefulness of two novel assays, namely the iTACT-hepatitis B surface antigen (iTACT-HBsAg) and iTACT-hepatitis B core-related antigen (iTACT-HBcrAg) assays, in chronic hepatitis B (CHB) patients with HBsAg seroclearance (SC) documented by standard assays. METHODS: HBsAg and HBcrAg were measured by the two iTACT-assays in 556 serial sera collected from 96 CHB patients at 7 different time points spanning from 5 years before to 10 years after SC and 120 HBsAg-negative, anti-HBc-positive individuals. As controls, 60 seronegative individuals, who were negative for HBsAg, anti-HBc and anti-HBs, were tested. RESULTS: Using the iTACT-assays, HBsAg was detectable in 154/418 (36.8%) samples collected after SC. HBcrAg was detectable in 78.3% and 65.9% of samples collected before and after SC, respectively. The detectability rates of both HBsAg and HBcrAg progressively decreased over time after SC. At 10 years after SC, 20.4% and 64.5% of the patients still had detectable HBsAg and HBcrAg, respectively. 66 (71%) patients had detectable HBsAg and/or HBcrAg. Among the 120 HBsAg-negative, anti-HBc-positive individuals, 11 (9.2%) and 4 (3.3%) had detectable HBsAg and HBcrAg respectively. Both HBsAg and HBcrAg were undetectable in the controls. CONCLUSION: The iTACT assays detected a low level of HBsAg and/or HBcrAg in >70% of patients even at 10 years after SC, suggesting that CHB patients with SC still harbour a low level of HBV protein expression. The clinical significance of detectable viral proteins after SC with regard to disease progression and HBV reactivation deserves further investigations.

Relationship between Uveal Inflammation and Viral Detection in 30 Cats with Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) virus is the most common infectious cause of uveitis in cats. Confirmatory diagnosis is usually only reached at postmortem examination. The relationship between the histologic inflammatory pattern, which depends on the stage of the disease, and the likelihood of detection of the viral antigen and/or RNA has not been investigated. We hypothesized that viral detection rate by either immunohistochemistry, in situ hybridization or RT-qPCR is dependent upon the predominant type of uveal inflammatory response (i.e., pyogranulomatous vs. plasmacytic). Thus, the aims of this study were to evaluate cases of FIP-induced uveitis, localize the viral antigen and RNA, and assess the relationship between the inflammatory pattern (macrophage- vs. plasma cell-rich) and the likelihood of detecting the FIP antigen and/or RNA. We evaluated 30 cats with FIP-induced uveitis. The viral antigen and/or RNA were detected within uveal macrophages in 11/30 cases, of which 8 tested positive by RT-qPCR. Correlation analysis determined a weak to moderate but significant negative correlation between the degree of plasmacytic uveal inflammation and the likelihood of detecting the FIP antigen and RNA. This study suggests that predominance of plasmacytic inflammation in cases of FIP uveitis reduces the odds of a confirmatory diagnosis through the viral detection methods available.