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Highly sensitive viral analytical techniques are essential tools for preventing the spread of infections. In this study, we established a digital enzyme-linked immunosorbent assay (ELISA) system to quantify norovirus proteins with high sensitivity. We used norovirus-like particles (noro-VLPs) as a surrogate for norovirus and constructed two digital ELISA systems using two different antibody pairs. The quantitative performance of the noro-VLP measurement using each digital ELISA system was evaluated. Both assay systems exhibited high sensitivity, good linearity, and high stability. The first system exhibited a limit of detection (LOD) of 87 pg mL -1 and correlation coefficient ( R 2 ) of 0.9984. Analysis of samples containing 5 ng per mL noro-VLP confirmed inter-assay variation of 5.5%, and intra-assay variation of 5.2%. The second system exhibited an LOD of 19 pg mL -1 and R 2 of 0.9984. Analysis of samples containing 5 ng per mL noro-VLP confirmed inter-assay variation of 4.5%, and intra-assay variation of 2.5%. Comparison of the two systems using the same calibrant for unpurified and fractionated noro-VLPs revealed that the quantitative values for unpurified noro-VLPs were the same, whereas those for fractionated noro-VLPs were dramatically different. Our findings indicate that the reactivity to various components in the noro-VLP solution was altered depending on the different antibodies. Furthermore, our study highlights the importance of using appropriate calibrants, which contain the same ratio of components as the noro-VLP analyte, to afford accurate measurements.

The COVID-19 pandemic has disproportionately affected workers in certain industries and occupations, and the workplace can be a high-risk setting for SARS-CoV-2 transmission. In this study, we measured SARS-CoV-2 antibody prevalence and identified work-related risk factors in a population primarily working at industrial livestock operations. We used a multiplex salivary SARS-CoV-2 IgG assay to determine infection-induced antibody prevalence among 236 adult (≥18 yr) North Carolina residents between February 2021 and August 2022. We used the National Institute for Occupational Safety and Health Industry and Occupation Computerized Coding System (NIOCCS) to classify employed participants' industry. Most participants (55%, 95% confidence interval [CI] 49% to 62%) were infection-induced IgG positive, including 71% (95% CI 60% to 83%) of animal slaughtering and processing industry workers, 1.5 to 4.3 times North Carolina general population infection-induced seroprevalence estimates during overlapping time periods. Considering self-reported diagnostic test positivity and vaccination history in addition to antibodies, the proportion of participants with evidence of prior infection increased slightly to 61% (95% CI 55% to 67%), including 75% (95% CI 64% to 87%) of animal slaughtering and processing workers. Participants with more than 1000 compared to 10 or fewer coworkers at their jobsite had higher odds of prior infection (adjusted odds ratio 4.5, 95% CI 1.0 to 21.0). This study contributes evidence of the severe and disproportionate impacts of COVID-19 on animal slaughtering and processing workers and workers in large congregate settings., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.)

On 19 January 2020, the first case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was identified in the United States, with the first cases in South Carolina confirmed on 06 March 2020. Due to initial limited testing capabilities and potential for asymptomatic transmission, it is possible that SARS-CoV-2 may have been present earlier than previously thought, while the immune status of at-risk populations was unknown. Saliva from 55 South Carolina emergency healthcare workers (EHCWs) was collected from September 2019 to March 2020, pre- and post-healthcare shifts, and stored frozen. To determine the presence of SARS-CoV-2-reactive antibodies, saliva-acquired post-shift was analysed by enzyme-linked immunosorbent assay (ELISA) with a repeat of positive or inconclusive results and follow-up testing of pre-shift samples. Two participants were positive for SARS-CoV-2 N/S1-reactive IgG, confirmed by follow-up testing, with S1 receptor binding domain (RBD)-specific IgG present in one individual. Positive samples were collected from medical students working in emergency medical services (EMSs) in October or November 2019. The presence of detectable anti-SARS-CoV-2 antibodies in 2019 suggests that immune responses to the virus existed in South Carolina, and the United States, in a small percentage of EHCWs prior to the earliest documented coronavirus disease 2019 (COVID-19) cases. These findings suggest the feasibility of saliva as a noninvasive tool for surveillance of emerging outbreaks, and EHCWs represent a high-risk population that should be the focus of infectious disease surveillance.

Background: Although dromedary camels (Camelus dromedarius) are known to be the host reservoir for MERS-CoV, the virus causing Middle East respiratory syndrome (MERS), zoonotic transmission pathways and camel subpopulations posing highest transmission risk are poorly understood. Extensively managed herds, ubiquitous across the Arabian Peninsula, present a major potential source of primary infection. In this study we aimed to address key knowledge gaps regarding MERS epidemiology among high-risk communities associated with such herds, which is essential information for effective control strategies., Methods: We did a cross-sectional study between Sept 27, 2017, and Oct 11, 2018, among members of livestock-owning households in southern Jordan (Aqaba East, Aqaba West, Ma'an East, and Ma'an West regions), with random selection of households (house and tent dwellings) from Ministry of Agriculture lists via computer-generated randomisation lists. Household visits were done, with questionnaires administered to household members regarding potential risk factors for MERS-CoV exposure in the past 6 months and blood samples and nasal and oral swabs collected, alongside physical examination data including blood pressure and blood glucose. Children younger than 5 years and individuals without capacity to provide informed consent were excluded. Serum was tested for IgG antibodies to MERS-CoV spike protein (S1 subunit) and nucleocapsid (N) protein with in-house indirect ELISAs, and viral RNA was detected in nasal and oral samples by RT-PCR. The primary outcome was evidence of MERS-CoV exposure (ascertained by seropositive status on S1 or N ELISAs, or a positive swab sample on RT-PCR); secondary outcomes were potential associations between possible risk factors and seropositive status. RT-PCR data were to be presented descriptively. Seroprevalence estimates were obtained at the individual and household levels, and associations between hypothetical risk factors and seropositive status were assessed with use of mixed-effects logistic regression., Findings: We sampled 879 household members (median age 27 years [IQR 16-44]; 471 [54%] males and 408 [46%] females) from 204 households. 72 (8%) household members were seropositive on S1 ELISA (n=25, 3%) or N ELISA (n=52, 6%). No positive nasal or oral swab samples were identified on RT-PCR. Within-household clustering was identified for seropositivity on S1 ELISA (intraclass correlation coefficient 0·88 [0·35-0·96]) but not N ELISA (0·00 [0·00-0·27]). On multivariable analysis, S1 ELISA seropositivity was associated with frequently (≥weekly) interacting with young (age <1 year) camels (adjusted odds ratio [OR adj ] 3·85 [95% CI 1·41-11·61], p=0·011), with frequent kissing and petting (OR adj 4·56 [1·55-15·42], p=0·0074), and frequent feeding and watering (OR adj 4·97 [1·80-15·29], p=0·0027) of young camels identified as risk activities. Attending camel races (OR adj 3·73 [1·11-12·47], p=0·029), frequently feeding and watering camels of any age (OR adj 3·18 [1·12-10·84], p=0·040), and elevated blood glucose (>150 mg/dL; OR adj 4·59 [1·23-18·36], p=0·021) were also associated with S1 ELISA seropositivity. Among individuals without history of camel contact, S1 ELISA seropositivity was associated with sharing a household with an S1 ELISA-positive household member (OR adj 8·92 [1·06-92·99], p=0·044), and with sharing a household with an S1 ELISA-positive household member with history of camel contact (OR adj 24·74 [2·72-306·14], p=0·0050). N ELISA seropositivity was associated with age (categorical, p=0·0069), a household owning a young camel (age <18 months; OR adj 1·98 [1·02-4·09], p=0·043), and frequently feeding and watering camels of any age (OR adj 1·98 [1·09-3·69]; p=0·025)., Interpretation: The study findings highlight the importance of effective MERS-CoV surveillance and control strategies among camel-owning communities in Jordan and the Arabian Peninsula. Juvenile dromedaries pose increased risk for zoonotic MERS-CoV transmission and should be prioritised for vaccination once such vaccines become available. Among high-risk communities, vaccination strategies should prioritise camel-owning households, particularly individuals engaged in camel husbandry or racing, and household members who are older or diabetic, with evidence to suggest secondary within-household transmission., Funding: UK Medical Research Council and US National Institute of Allergy and Infectious Diseases., Competing Interests: Declaration of interests EAA-B was project coordinator for the project “Reducing the threat of MERS-CoV and avian influenza in Jordan and strengthening regional disease surveillance capacity”, supported by the US Department of Defense, Defense Threat Reduction Agency, Biological Threat Reduction Program. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The SARS-CoV-2 outbreak has provoked more than 6 million deaths worldwide. The scarcity of effective treatments and its virulence converted the vaccines into an essential tool to face it. The most used vaccines were the mRNA, adenovirus vector, and inactivated whole-virus. However, nowadays, infants aged < 6 months are not eligible for any vaccines against COVID-19, and their immunization relies on passive immunity. In this research, we investigated the humoral and cellular immune response generated on newborns of SARS-CoV-2 vaccinated mothers with mRNA or viral vector (VV) vaccine employing Fourier transformed infrared (FTIR) spectroscopy in saliva samples. For this purpose, saliva samples of newborns and their mothers were collected; the population was divided into two groups, VV and mRNA, which were subdivided into three subgroups: before pregnancy (BP), at the first (FTP) and second (STP) trimesters of pregnancy. The samples were analyzed using FTIR spectroscopy, and the bands associated with the humoral and cellular immune responses, such as IgG, IgA, and IFN-γ were analyzed. The integrated areas were calculated and compared to elucidate the quantity of those immunoglobins and the cytokine. Likewise, the correlation of the humoral and cellular immune response between the newborns and their mothers and the correlation between cellular and humoral immune response was also evaluated. The VV vaccine produced a significant humoral and cellular immune response in newborns and their mothers when they received it at the STP compared with the mRNA vaccine, evidencing statistical significance. However, no correlation was observed between newborns and their mothers when the vaccine was applied in this trimester of pregnancy. When administered BP, the mRNA vaccine generated more humoral immunity in newborns and their mothers. Nevertheless, compared with the VV vaccine, it only showed statistical significance in the mothers, highlighting that IgG showed a moderate positive correlation between the newborns and their mothers., (© 2024. The Author(s).)

Dengue virus (DENV) is the most prevalent global arbovirus, exhibiting a high worldwide incidence with intensified severity of symptoms and alarming mortality rates. Faced with the limitations of diagnostic methods, an optical and electrochemical biosystem was developed for the detection of DENV genotypes 1 and 2, using cysteine (Cys), cadmium telluride (CdTe) quantum dots, and anti-DENV antibodies. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), surface plasmon resonance (SPR), atomic force microscopy (AFM), and Fourier transform infrared spectroscopy (FTIR) were employed to characterize the immunosensor. The AFM and SPR results demonstrated discernible topographic and angular changes confirming the biomolecular recognition. Different concentrations of DENV-1 and DENV-2 were evaluated (0.05 × 10 6 to 2.0 × 10 6 PFU mL -1 ), resulting in a maximum anodic shift (Δ I %) of 263.67% ± 12.54 for DENV-1 and 63.36% ± 3.68 for DENV-2. The detection strategies exhibited a linear response to the increase in viral concentration. Excellent linear correlations, with R 2 values of 0.95391 for DENV-1 and 0.97773 for DENV-2, were obtained across a broad concentration range. Data analysis demonstrated high reproducibility, displaying relative standard deviation values of 3.42% and 3.62% for Cys-CdTe-antibody DENV-1 -BSA and Cys-CdTe-antibody DENV-2 -BSA systems. The detection limits were 0.34 × 10 6 PFU mL -1 and 0.02 × 10 6 PFU mL -1 , while the quantification limits were set at 1.49 × 10 6 PFU mL -1 and 0.06 × 10 6 PFU mL -1 for DENV-1 and DENV-2, respectively. Therefore, the biosensing apparatus demonstrates analytical effectiveness in viral screening and can be considered an innovative solution for early dengue diagnosis, contributing to global public health.

Background: The coronavirus disease 2019 (COVID-19) pandemic offered an epidemiological opportunity to evaluate if isolation and masking affected John Cunningham (JC) virus transmission., Objective: This study aimed to assess the proportion of natalizumab-treated patients who converted to a positive anti-JCV antibody serostatus before and during the pandemic., Methods: Data from TYSABRI Outreach: Unified Commitment to Health (TOUCH) for 22,375 US patients treated with natalizumab with anti-JCV antibody records were assessed in epochs annually from 2017 to 2022., Results: Pre-pandemic anti-JCV antibody serostatus change was observed for 7.4%-7.7%. During the first and second years of the pandemic, 7.3% and 7.2% of patients' serostatus changed, respectively., Conclusion: The proportion of patients with anti-JCV antibody serostatus change did not significantly differ during the first 2 years of the pandemic compared with prior years. In contrast to seasonal influenza, masking and social distancing had no discernable effect on JCV serostatus change., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: S.C.K. reports consulting or advisory work with Biogen, EMD Serono, Genentech, Genzyme/Sanofi, Novartis, Octave, and TG Therapeutics; nonpromotional speaking with Biogen, EMD Serono, and Genentech; grant and research support from Biogen, Bristol Myers Squibb (BMS), Novartis, and Sanofi. S.S., F.H., J.S., T.J.K., K.W., and R.L.A. are the employees of and may hold stock and/or stock options in Biogen.

Mucosal antibodies in the upper respiratory tract are the earliest and most critical responders to prevent respiratory infections, providing an indication for the rapid evaluation of immune protection. Here, we report a microfluidic particle counter that directly visualizes mucosal antibody levels in nasal mucus. The mucosal anti-SARS-CoV-2 spike receptor binding domain (RBD) antibodies in nasal secretions first react with magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that are surface-modified to form a "MMPs-anti-spike RBD IgG-PMPs" complex when RBD is present. After magnetic separation and loading into the microfluidic particle counter, the free PMPs, which are reduced with increasing anti-spike RBD IgG antibody levels, are trapped by a microfluidic particle dam and accumulate in the trapping channel. A sensitive mode [limit of detection (LOD): 14.0 ng mL -1 ; sample-to-answer time: 70 min] and an equipment-free rapid mode (LOD: 37.4 ng mL -1 ; sample-to-answer time: 20 min) were achieved. Eighty-seven nasal secretion (NS) samples from vaccinees were analyzed using our microfluidic particle counter, and the results closely resemble those of the gold-standard enzyme-linked immunosorbent assay (ELISA). The analysis shows that higher antibody levels were found in convalescent volunteers compared to noninfected volunteers. Together, we demonstrate a rapid kit that directly indicates immune status, which can guide vaccine strategy for individuals and the government.

Objective: Newborns are vulnerable to all types of infections due to their developing immune system. To compensate for their immune immaturity, newborns rely on the passive transfer of antibodies through the placenta and own mother's breast milk (BM). In the present study, we investigated whether vaccination against SARS-CoV-2 leads to the presence of antibodies in BM. Furthermore, we compared the levels of SARS-CoV-2-specific anti-spike (anti-S) IgG antibodies in the BM of mothers who were vaccinated against Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or had coronavirus disease 2019 (COVID-19) infection naturally or were vaccinated after natural infection., Study Design: This was a prospective cohort study conducted in the Ondokuz Mayis University Faculty of Medicine. Forty-six mothers who had at least two doses of the BNT162b2 messenger RNA-based vaccine and/or had a history of symptomatic COVID-19 infection were included in the study. BM samples were analyzed by the Abbott Architect SARS-CoV-2 IgG II Quant kit following the manufacturer's instructions., Results: Forty-six mothers with an average age of 29.7 ± 5.7 years participated the study: 18 (39.1%) had COVID-19 infection + BTN162b2 vaccine, 17 (37.0%) had BTN162b2 vaccine, and 11 (23.9%) had natural infection. The highest SARS-CoV-2-specific anti-S IgG antibody titers in BM were found in mothers who were vaccinated following the infection (anti-SARS-CoV-2 IgG: 32.48 ± 57.1 arbitrary units AU/mL). However, no significant difference in anti-SARS-CoV-2 antibody levels was observed between the three groups ( p  = 0.641). No antibody was detected in 15.2% of BM samples., Conclusion: Both vaccination and natural COVID-19 infection during pregnancy leads to the passive transfer of specific anti-SARS-CoV-2 IgG antibodies to BM. These results are important to overcome vaccine hesitancy and increase vaccination levels in expectant mothers., Key Points: · We investigated the levels of SARS-CoV-2 antibodies in BM after natural infection and vaccination.. · Anti-SARS-CoV-2 IgG antibodies were detected in 39 (84.8%) BM samples.. · The highest titers in BM were found in mothers who were vaccinated following natural infection.., Competing Interests: None declared., (Thieme. All rights reserved.)

Objectives: To investigate specific immunoglobulin A (sIgA), specific immunoglobulin G (sIgG), and neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in breast milk and compare immunity in mothers with hybrid immunity (infection and vaccination) versus those solely vaccinated (coronavirus disease [COVID]-naïve). Methods: A longitudinal study was conducted among lactating mothers who received at least two doses of the coronavirus disease 2019 (COVID-19) vaccine or tested positive for SARS-CoV-2. Details of vaccination and infection were collected through questionnaires and interviews. Fifteen milliliters of breast milk samples, self-collected at 1, 3, and 6 months postvaccination or infection, were sent to analysis for sIgA, sIgG, and NAbs using enzyme-linked immunosorbent assay. Results: In total, 119 lactating mothers (202 milk samples) were enrolled; 82 participants had hybrid immunity, and 32 were COVID-19-naïve. Two-thirds received a combination of different vaccines and booster shots. Breast milk retained sIgA, sIgG, and NAbs for up to 6 months post-COVID vaccination or infection. At 3 months, mothers with hybrid immunity had significantly higher sIgA and NAbs compared with COVID-naïve mothers (geometric mean [95% confidence interval (CI)] of sIgA 2.72 [1.94-3.8] vs. 1.44 [0.83-2.48]; NAbs 86.83 [84.9-88.8] vs. 81.28 [76.02-86.9]). No differences in sIgA, sIgG, and NAbs were observed between lactating mothers receiving two, three, or more than or equal to three doses, regardless of hybrid immunity or COVID-naïve status. Conclusion: sIgA, sIgG, and NAbs against SARS-CoV-2 in breast milk sustained for up to 6 months postimmunization and infection. Higher immunity was found in mothers with hybrid immunity. These transferred immunities confirm in vitro protection, supporting the safety of breastfeeding during and after COVID-19 vaccination or infection.

Outbreaks of viral diseases seriously jeopardize people's health and cause huge economic losses. At the same time, virology provides a new perspective for biology, molecular biology and cancer research, and it is important to study the discovered viruses with potential applications. Therefore, the development of immediate and rapid viral detection methods for the prevention and treatment of viral diseases as well as the study of viruses has attracted extensive attention from scientists. With the continuous progress of science and technology, especially in the field of bioanalysis, a series of new detection techniques have been applied to the on-site rapid detection of viruses, which has become a powerful approach for human beings to fight against viruses. In this paper, the latest research progress of rapid point-of-care detection of viral nucleic acids, antigens and antibodies is presented. In addition, the advantages and disadvantages of these technologies are discussed from the perspective of practical application requirements. Finally, the problems and challenges faced by rapid viral detection methods and their development prospects are discussed.

Importance: There is still considerable controversy in the literature regarding the capacity of intramuscular messenger RNA (mRNA) vaccination to induce a mucosal immune response., Objective: To compare serum and salivary IgG and IgA levels among mRNA-vaccinated individuals with or without previous SARS-CoV-2 infection., Design, Setting, and Participants: In this cohort study, SARS-CoV-2-naive participants and those with previous infection were consecutively included in the CoviCompare P and CoviCompare M mRNA vaccination trials and followed up to day 180 after vaccination with either the BNT162b2 (Pfizer-BioNTech) vaccine or the mRNA-1273 (Moderna) vaccine at the beginning of the COVID-19 vaccination campaign (from February 19 to June 8, 2021) in France. Data were analyzed from October 25, 2022, to July 13, 2023., Main Outcomes and Measures: An ultrasensitive digital enzyme-linked immunosorbent assay was used for the comparison of SARS-CoV-2 spike-specific serum and salivary IgG and IgA levels. Spike-specific secretory IgA level was also quantified at selected times., Results: A total of 427 individuals were included in 3 groups: participants with SARS-CoV-2 prior to vaccination who received 1 single dose of BNT162b2 (Pfizer-BioNTech) (n = 120) and SARS-CoV-2-naive individuals who received 2 doses of mRNA-1273 (Moderna) (n = 172) or 2 doses of BNT162b2 (Pfizer-BioNTech) (n = 135). The median age was 68 (IQR, 39-75) years, and 228 (53.4%) were men. SARS-CoV-2 spike-specific IgG saliva levels increased after 1 or 2 vaccine injections in individuals with previous infection and SARS-CoV-2-naive individuals. After vaccination, SARS-CoV-2-specific saliva IgA levels, normalized with respect to total IgA levels, were significantly higher in participants with previous infection, as compared with the most responsive mRNA-1273 (Moderna) recipients (median normalized levels, 155 × 10-5 vs 37 × 10-5 at day 29; 107 × 10-5 vs 54 × 10-5 at day 57; and 104 × 10-5 vs 70 × 10-5 at day 180 [P < .001]). In contrast, compared with day 1, spike-specific IgA levels in the BNT162b2-vaccinated SARS-CoV-2-naive group increased only at day 57 (36 × 10-5 vs 49 × 10-5 [P = .01]). Bona fide multimeric secretory IgA levels were significantly higher in individuals with previous infection compared with SARS-CoV-2-naive individuals after 2 antigenic stimulations (median optical density, 0.36 [IQR, 0.16-0.63] vs 0.16 [IQR, 0.10-0.22]; P < .001)., Conclusions and Relevance: The findings of this cohort study suggest that mRNA vaccination was associated with mucosal immunity in individuals without prior SARS-CoV-2 infection, but at much lower levels than in previously infected individuals. Further studies are needed to determine the association between specific saliva IgA levels and prevention of infection or transmission.

Vaccination against measles is one of the most effective public health interventions which has saved millions of lives and interrupted circulation of the natural virus in the population. However, it is widely accepted that the immunity after vaccination can wane, especially in those who have had no contact with the virus. This study aimed to classify the particular birth cohorts of adults with regard to their exposure to the wild measles virus in the population with a long history of mandatory vaccination. We introduced two methods. In the first, we estimated the probability of exposure to the wild virus through an analysis of antibody levels from the Immunologic Survey performed in the Slovak Republic in 2018, while the second was based on historical epidemiological data. Both methods resulted in similar estimations. Cohorts born in Slovakia before 1964 can be considered to be cohorts in which most people were exposed to the wild measles virus. Cohorts born after 1977 can be designated as cohorts that most likely did not come into the contact with the wild virus. Cohorts born between 1965 and 1976 are composed of a mixture, with a decreasing proportion of people exposed to the wild virus with increasing year of birth. The proposed methods can help identify potential immunity gaps in the adult population. They can be applied in other countries with high measles vaccination coverage to estimate the probability of exposure to the wild measles virus in particular birth cohorts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Influenza B viruses (IBV) cocirculate with influenza A viruses (IAV) and cause periodic epidemics of disease, yet antibody and cellular responses following IBV infection are less well understood. Using the ferret model for antisera generation for influenza surveillance purposes, IAV resulted in robust antibody responses following infection, whereas IBV required an additional booster dose, over 85% of the time, to generate equivalent antibody titers. In this study, we utilized primary differentiated ferret nasal epithelial cells (FNECs) which were inoculated with IAV and IBV to study differences in innate immune responses which may result in differences in adaptive immune responses in the host. FNECs were inoculated with IAV (H1N1pdm09 and H3N2 subtypes) or IBV (B/Victoria and B/Yamagata lineages) and assessed for 72 h. Cells were analyzed for gene expression by quantitative real-time PCR, and apical and basolateral supernatants were assessed for virus kinetics and interferon (IFN), respectively. Similar virus kinetics were observed with IAV and IBV in FNECs. A comparison of gene expression and protein secretion profiles demonstrated that IBV-inoculated FNEC expressed delayed type-I/II IFN responses and reduced type-III IFN secretion compared to IAV-inoculated cells. Concurrently, gene expression of Thymic Stromal Lymphopoietin ( TSLP ), a type-III IFN-induced gene that enhances adaptive immune responses, was significantly downregulated in IBV-inoculated FNECs. Significant differences in other proinflammatory and adaptive genes were suppressed and delayed following IBV inoculation. Following IBV infection, ex vivo cell cultures derived from the ferret upper respiratory tract exhibited reduced and delayed innate responses which may contribute to reduced antibody responses in vivo .IMPORTANCEInfluenza B viruses (IBV) represent nearly one-quarter of all human influenza cases and are responsible for significant clinical and socioeconomic impacts but do not pose the same pandemic risks as influenza A viruses (IAV) and have thus received much less attention. IBV accounts for greater severity and deaths in children, and vaccine efficacy remains low. The ferret can be readily infected with human clinical isolates and demonstrates a similar course of disease and immune responses. IBV, however, generates lower antibodies in ferrets than IAV following the challenge. To determine whether differences in initial innate responses following infection may affect the development of robust adaptive immune responses, ferret respiratory tract cells were isolated, infected with IAV/IBV, and compared. Understanding the differences in the initial innate immune responses to IAV and IBV may be important in the development of more effective vaccines and interventions to generate more robust protective immune responses., Competing Interests: The authors declare no conflict of interest.

This study explores the correlation between immunological and clinical characteristics in coronavirus disease 2019 (COVID-19) patients with detectable severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in feces, analyzing data from 251 patients admitted to Mostar University Clinical Hospital (UCH) from December 2021 to January 2022. Methods involved reverse transcription quantitative polymerase chain reaction (RT-qPCR) from nasopharyngeal (NP) swabs and feces, alongside serological tests for anti-SARS-CoV-2 spike IgGs. Demographic and clinical data were collected through questionnaires and medical records. The data analyses were performed using SPSS statistical software. Death occurred in 53 patients (21.1%, P < 0.001), mostly in the elderly (47/53, 88.7%, P = 0.001) and immunocompromised (19/53, 35.8%, P = 0.05), particularly those developing acute respiratory insufficiency (ARI) (46/53, 86.8%, P = 0.004), and severe/critical disease (46/53, 86.8%, P = 0.002). Among the patients with positive anti-SARS-CoV-2 IgG antibodies (86/251, 34.3%, P < 0.001), 41 (47.7%) were vaccinated and 45 (52.3%) unvaccinated (P = 0.666), showing no significant differences in clinical outcomes or mortality. Unvaccinated patients with a negative antibody titer had a higher incidence of ARI (96/123, 78%, P = 0.029) and intensive care unit (ICU) admission (22/123, 17.9%, P = 0.026), than those with a positive antibody titer. Forty-seven (62.7%) patients, out of the 75 hospitalized who provided a feces sample, were positive for SARS-CoV-2 RNA (P = 0.028), without statistical differences between fecal SARS-CoV-2 positive and negative groups regarding vaccination status (15/47, 31.9%, P = 0.493), antibody status (18/47, 38.3%, P = 0.628), or death outcome (5/47, 10.6%, P = 0.706). In conclusion, unvaccinated hospitalized patients with a severe COVID-19 presentation and a negative anti-spike SARS-CoV-2 IgG titer had adverse outcomes more frequently. This suggests cautious consideration for the diagnostic use of fecal samples compared to NP swabs.

The most preferred method for the detection of foot-and-mouth disease (FMD) viral antigen and identification of viral serotype is the enzyme-linked immunosorbent assay (ELISA). Diagnostic tests with high sensitivity are necessary both to distinguish infected vaccinated animals and execute disease control programs for the identification of the carrier animals. The current strategies for the detection of FMD virus are mainly based on the capture antibody (sandwich) ELISA test. The usage of laying pullets as an animal bioreactor for the production of specific egg yolk antibodies (IgY) has increased in recent years due to its high yield, affinity, low price, and quick production turnover. The present study aimed to produce a concentrated and purified IgY polyclonal antibody to design a capture antibody ELISA kit against the FMD virus (FMDV) serotype A. At first, laying hens were immunized with inactivated FMDV serotype virus, and then, on days 14, 21, and 28 following vaccination, the eggs and sera were collected. Afterward, the IgY polyclonal antibodies were extracted and purified from the chicken egg yolk using a polyethylene glycol 6000-ethanol precipitation procedure. Extracts were filtered, purified by ion exchange chromatography, and dialyzed. The purified IgY concentration, estimated by Bradford assay, confirmed its presence by SDS-PAGE and Western blot and also its specific immune reaction by Ouchterlony double immunodiffusion and Dot blot tests. Moreover, for achieving the optimum concentration of antigen/antibody (sera) in sandwich ELISA, a checkerboard titration test was set up based on indirect ELISA results. Eventually, 119 previously confirmed samples (including 80 positive and 39 negative) by both real-time polymerase chain reaction (quantitative PCR, qPCR) and a commercial ELISA kit were used for evaluation of the sensitivity and accuracy of our developed Capture antibody ELISA kit. In this manner, the sensitivity and specificity of our designed kit were 100% and 98%, respectively. Accordingly, the present developed capture ELISA kit based on IgY had high sensitivity and specificity for FMD virus detection and it could be used in the future for both commercial detecting and serotyping applications., Competing Interests: The authors declare that there is no conflict of interest.

Background: While the presence of SARS-CoV-2 in human breast milk is contentious, anti-SARS-CoV-2 antibodies have been consistently detected in human breast milk. However, it is uncertain when and how long the antibodies are present., Methods: This was a prospective cohort study including all consecutive pregnant women with confirmed SARS-CoV-2 infection during pregnancy, recruited at six maternity units in Spain and Hong Kong from March 2020 to March 2021. Colostrum (day of birth until day 4 postpartum) and mature milk (day 7 postpartum until 6 weeks postpartum) were prospectively collected, and paired maternal blood samples were also collected. Colostrum samples were tested with rRT-PCR-SARS-CoV-2, and skimmed acellular milk and maternal sera were tested against SARS-CoV-2 specific immunoglobulin M, A, and G reactive to receptor binding domain of SARS-CoV-2 spike protein 1 to determine the presence of immunoglobulins. Then, we examined how each immunoglobulin type in the colostrum was related to the time of infection by logistic regression analysis, the concordance between these immunoglobulins in the colostrum, maternal serum, and mature milk by Cohen's kappa statistic, and the relationship between immunoglobulin levels in mature milk and colostrum with McNemar., Results: One hundred eighty-seven pregnant women with confirmed SARS-CoV-2 infection during pregnancy or childbirth were recruited and donated the milk and blood samples. No SARS-CoV-2 was found in the human breast milk. Immunoglobulin A, G, and M were present in 129/162 (79·6%), 5/163 (3·1%), and 15/76 (19·7%) colostrum samples and in 17/62 (27·42%), 2/62 (3·23%) and 2/62 (3·23%) mature milk samples, respectively. Immunoglobulin A was the predominant immunoglobulin found in breast milk, and its levels were significantly higher in the colostrum than in the mature milk (p-value < 0.001). We did not find that the presence of immunoglobulins in the colostrum was associated with their presence in maternal, the severity of the disease, or the time when the infection had occurred., Conclusions: Since anti-SARS-CoV-2 antibodies are found in the colostrum irrespective of the time of infection during pregnancy, but the virus itself is not detected in human breast milk, our study found no indications to withhold breastfeeding, taking contact precautions when there is active disease., (© 2024. The Author(s).)

Mumps reemergence has been reported in developed countries with high levels of two-dose mumps-containing vaccine (MuCV) coverage. The effectiveness of the two-dose MuCV may be compromised by limitations in the persistence of immunity. This prospective cohort study evaluated the persistence of immunity of a two-dose MuCV in children aged 3-7 years from 2015 to 2020. Persistence of antibody to mumps, determined as the geometric mean antibody concentration (GMC), and seropositivity were analyzed for both repeated measurements from three follow-ups and on each cross-section, respectively. A total of 105 eligible subjects were recruited. Their overall seropositivity rate was relatively high and stable (92.4%-84.8%), while the overall GMC decreased from 547.6 U/ml to 333.3 U/ml. Analysis of waning immunity in 91 participants showed a significant and consistent downward trend for GMC, which differed significantly in boys and girls. The overall seropositivity rate decreased slightly from 2015 (95.6%) to 2016 (92.3%) but both were significantly higher than in 2018 (84.6%). The rates in girls remained stable, while those in boys declined to 75% in 2018. The seropositivity rate of the cross-section level decreased from 95.4% to 86.4% in 4 years. Although two-dose MuCV may result in a high level of immunity, antibody concentrations decay over 2 years after the second dose. Children with waning immunity after receiving two doses, especially boys, require further surveillance at 4 years and later to avoid future mumps epidemics. Clinical trial registration: NCT02901990.

Objective: The purpose of this case study was to describe the transmission of porcine reproductive and respiratory syndrome virus (PRRSV) under field and experimental conditions via the consumption of PRRSV-positive swine feed., Animals: 1 domestic swine breeding herd and 20 laboratory-maintained experimental domestic pigs., Clinical Presentation, Progression, and Procedures: A 2,500-sow PRRSV-naïve biosecure breeding herd became infected during the autumn months. It experienced a feed outage involving a specific bin on October 23 (day 0), with the bin refilled on October 24 (day 1). From October 28 to 30 (days 5 to 7), signs of anorexia and hyperemia were observed in 30 gestating sows after consuming feed from this bin. On November 1 (day 9), blood samples from 10 affected sows were PRRSV positive by reverse transcriptase PCR. In contrast, sows in the same room that had consumed feed from other bins were clinically normal and PRRSV negative. To investigate whether the feed delivery introduced PRRSV to the herd, on November 2 (day 10) 4 samples of feed material from the interior walls of the index bin were collected and tested by reverse transcriptase PCR., Treatment and Outcome: All 4 samples were positive for PRRSV RNA with cycle threshold values ranging from 26 to 29. Nucleic acid sequencing indicated that the open reading frame 5 region of the PRRSV in feed samples was 100% homologous to PRRSV from index cases. To assess viability of the virus, PRRSV-naïve pigs were allowed to consume the index feed bin samples and became infected with PRRSV based on viral RNA in oral fluid samples, clinical signs, and postmortem lesions., Clinical Relevance: These results suggest that feed was a likely source of PRRSV introduction to the herd. This is the first report of PRRSV transmission through feed.

Bovine ephemeral fever virus (BEFV) is a globally spread arthropod-borne RNA virus that has significant economic impacts on the cattle industry. A live attenuated commercial BEF vaccine, based on the Australian BEFV strain 919, is widely used in Israel and other countries. A previous study has suggested the high effectiveness of this vaccine (ULTRAVAC BEF VACCINE™ from Zoetis®), but anecdotal reports of high BEF morbidity among vaccinated dairy herds in Israel casted doubt on these findings. To resolve this uncertainty, a randomized controlled field vaccine effectiveness study was conducted in Israel during a BEF outbreak which occurred in 2021. Eleven dairy herds were enrolled and monitored for BEF-associated morbidity and rumination alteration patterns using electronic monitoring tags (HR Tags, SCR® Dairy, Netanya, Israel). Four of the herds were naturally infected with BEFV during the outbreak, resulting in a total of 120 vaccinated and 311 unvaccinated subjects that were included in the effectiveness study. A mixed-effect Cox proportional hazard regression model was used to calculate the overall hazard ratio between vaccinated and unvaccinated cattle. This analysis demonstrated an average vaccine effectiveness of 60 % (95 % CI = 38 %-77 %) for preventing clinical disease. In addition, a non-statistically significant trend (p = 0.1) towards protection from mortality was observed, with no observation of mortality among the vaccinated groups compared to 2.61 % mortality (7/311) among the unvaccinated subjects. One hundred and thirty vaccinated and unvaccinated calves from affected and non-affected herds and with different status of morbidity were sampled and analysed by serum-neutralization test. The highest titers of BEFV-neutralizing antibodies were found in subjects that were both vaccinated and clinically affected, indicating a booster effect after vaccination. The results of the study provide evidence for the moderate effectiveness of the ULTRAVAC BEF VACCINE™ for the prevention of BEF., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Eyal Klement reports financial support was provided by Israel Ministry of Agriculture and Rural Development., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Introduction: Intravenous immunoglobulin (IVIG) preparations, used for the treatment of antibody deficiencies, provide a glimpse of the general population's antibody profile as each preparation is generated from a pool of thousands of donors. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the coronavirus disease 2019 (Covid-19) pandemic, and a vaccine for the prevention of Covid-19 was authorized for emergency use in December 2020. We completed a longitudinal analysis of SARS-CoV-2 antibody levels in commercial IVIG preparations., Materials and Methods: We collected IVIG samples from our infusion clinic. IVIG product lot number, product name, and manufacturer information were recorded, with the date of preparation verified from the manufacturer. SARS-CoV-2 antibody titers as well as total immunoglobulin levels were measured using commercially available assays. The study received Institutional Review Board approval., Results: We found no SARS-CoV-2 antibodies in preparations generated on or before January 2020. Overall, SARS-CoV-2 antibody levels in IVIG preparations tended to increase with progressing preparation date. We observed a dramatic and continual rise of SARS-CoV-2 antibody levels in IVIG preparations made in the beginning after January 2021, coinciding with the peak in incidence of confirmed cases and availability of Covid-19 vaccines in the United States., Conclusion: SARS-CoV-2 antibody levels in IVIG mirror case prevalence, and vaccination resulted in a far more rapid rate of rise in antibody levels. IVIG preparations or serum repositories can provide an accessible way to model a population's evolving novel pathogen exposure, immunity, and vaccine response., (Published by Oxford University Press on behalf of the Association of Military Surgeons of the United States 2022. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV). Vaccination is an effective approach for controlling IBV. Therefore, reliable immune monitoring for IB is critical for poultry. In this study, a novel peptide derived from S2 protein was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of broadly cross-reactive antibodies against IBV. The peptide-based ELISA (pELISA) showed good specificity and sensitivity in detecting IBV antibodies against different serotypes. A semilogarithmic regression method for determining IBV antibody titers was also established. Antibody titers detected by pELISA and calculated with this equation were statistically similar to those evaluated by indirect fluorescence assay (IFA). Moreover, the comparison analysis showed a 96.07% compatibility between the pELISA and IDEXX ELISA. All these data demonstrate that the pELISA generated here can be as a rapid and reliable serological surveillance tool for monitoring IBV infection or vaccination., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Detection of SARS-CoV-2 antibodies is essential in establishing the parameters of an individual's immune response to COVID-19, from both natural infection and vaccination. Despite this, there is currently limited clinical guidance or recommendations for serological methods for their measurement. Here, we evaluate and compare four Luminex-based assays for the multiplex detection of IgG SARS-CoV-2 antibodies., Methods: The four assays tested were Magnetic Luminex Assay, MULTICOV-AB Assay, Luminex xMAP SARS-CoV-2 Multi-Antigen IgG Assay and LABScreen COVID Plus Assay. Each assay's ability to detect antibodies to SARS-CoV-2 Spike (S), Nucleocapsid (N) and Spike-Receptor Binding Domain (RBD) was evaluated using 50 test samples (25 positive, 25 negative), previously tested by a widely used ELISA technique., Results: The MULTICOV-AB Assay had the highest clinical performance detecting antibodies to S trimer and RBD in 100% (n = 25) of known positive samples. Both the Magnetic Luminex Assay and LABScreen COVID Plus Assay showed significant diagnostic accuracy with sensitivities of 90% and 88% respectively. The Luminex xMAP SARS-CoV-2 Multi-Antigen IgG Assay demonstrated limited detection of antibodies to the S antigen resulting in a sensitivity of 68%., Conclusion: Luminex-based assays provide a suitable serological method for multiplex detection of SARS-CoV-2 specific antibodies, with each assay able to detect antibodies to a minimum of 3 different SARS-CoV-2 antigens. Assay comparison identified there is moderate performance variability between manufacturers and further inter-assay variation of antibodies detected to different SARS-CoV-2 antigens., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Coronavirus disease (COVID-19) has generated interest in the assessment of systemic immune status, but existing knowledge about mucosal immunity is clearly insufficient to understand the full pathogenetic mechanisms of the disease. The aim of this study was to evaluate the long-term effects of novel coronavirus infection on mucosal immunity in the postinfection period among health care workers (HCWs). A total of 180 health care workers with and without a history of COVID-19 who ranged in age from 18 to 65 years were enrolled in this one-stage, cross-sectional study. The study subjects completed the 36-Item Short Form (36) Health Survey (SF-36) and the Fatigue Assessment Scale. Secretory immunoglobulin A (sIgA) and total immunoglobulin G (IgG) levels were quantified in saliva samples, induced sputum samples, and nasopharyngeal and oropharyngeal scrapings by an enzyme-linked immunosorbent assay. Specific anti-SARS-CoV-2 IgG antibodies were quantified in serum samples by chemiluminescence immunoassay. Analysis of the questionnaire data showed that all HCWs with a history of COVID-19 reported health problems that limited their daily activities and negative changes in their emotional health three months after the disease, regardless of its severity. The following shifts were detected in the adaptive arm of the immune response in different mucosal compartments. Among subjects who had severe or moderate-to-severe COVID-19, salivary sIgA levels were significantly higher than those in the control group (p < 0.05 and p < 0.005, respectively). Compared to the subjects in the control group, all subjects with prior COVID-19 had significantly higher levels of total IgG in induced sputum. In the group of patients who had had severe infection, total IgG in saliva was also higher (p < 0.05). A direct statistically significant correlation was also detected between the levels of total IgG in all studied samples and the levels of specific IgG antibodies against SARS-CoV-2 in the serum. A significant correlation was observed between total IgG levels and the parameters of physical and social activities, mental health, and fatigue levels. Our study demonstrated long-term changes in the humoral mucosal immune response, which were most pronounced in health care workers with a history of severe or moderate-to-severe COVID-19, and an association of these changes with certain clinical signs of post-COVID-19 syndrome., (© 2023. The Author(s).)

A single-molecule assay (SiMoA) using a digital enzyme-linked immunosorbent assay (ELISA) has been attracting attention as a promising method that can detect viruses with ultra-high sensitivity. However, the quantitative application of digital ELISA has not been adequately reported. Therefore, in this study, we first evaluated the linearity and sensitivity of digital ELISA using a Certified Reference Material of C-reactive protein (NMIJ CRM 6201-c) as a quality control material. Next, we originally screened those antibody pair that are suitable for detecting recombinant viral proteins of influenza A virus, nucleoprotein (NP), and hemagglutinin (HA), and established the measurement system. Under optimized conditions, the limit of detection (LOD) of NP and HA was 0.59 fM and 0.99 fM, and the coefficient of determination, R 2 , was 0.9998 and 0.9979, respectively. Two subtypes of influenza virus, A/Puerto Rico/8/1934 (H1N1) [PR8] and A/Panama/2007/99 (H3N2) [Pan99], were also quantified under established conditions, and the LOD of PR8 was 3.1 × 10 2 PFU/mL on targeting NP and 7.4 × 10 2 PFU/mL on targeting HA. The LOD of Pan99 was 5.3 × 10 2 PFU/mL on targeting NP. The specificity and robustness of the recombinant viral protein and influenza virus measurements using digital ELISA were also evaluated. Our measurement system showed enough specificity to discriminate the viral subtypes properly and showed sufficient inter- and intra-assay variations for both measurements of recombinant viral proteins and viruses, except for NP-targeting virus measurement., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

Bovine Coronavirus (BCoV) is a major pathogen associated with neonatal calf diarrhea. Standard practice dictates that to prevent BCoV diarrhea, dams should be immunized in the last stage of pregnancy to increase BCoV-specific antibody (Ab) titers in serum and colostrum. For the prevention to be effective, calves need to suck maternal colostrum within the first six to twelve hours of life before gut closure to ensure a good level of passive immunity. The high rate of maternal Ab transfer failure resulting from this process posed the need to develop alternative local passive immunity strategies to strengthen the prevention and treatment of BCoV diarrhea. Immunoglobulin Y technology represents a promising tool to address this gap. In this study, 200 laying hens were immunized with BCoV to obtain spray-dried egg powder enriched in specific IgY Abs to BCoV on a large production scale. To ensure batch-to-batch product consistency, a potency assay was statistically validated. With a sample size of 241, the BCoV-specific IgY ELISA showed a sensitivity and specificity of 97.7% and 98.2%, respectively. ELISA IgY Abs to BCoV correlated with virus-neutralizing Ab titers (Pearson correlation, R 2 = 0.92, p < 0.001). Most importantly, a pilot efficacy study in newborn calves showed a significant delay and shorter duration of BCoV-associated diarrhea and shedding in IgY-treated colostrum-deprived calves. Calves were treated with milk supplemented with egg powder (final IgY Ab titer to BCoV ELISA = 512; VN = 32) for 14 days as a passive treatment before a challenge with BCoV and were compared to calves fed milk with no supplementation. This is the first study with proof of efficacy of a product based on egg powder manufactured at a scale that successfully prevents BCoV-associated neonatal calf diarrhea.

Background: Rapid IgM/IgG antibody tests were largely used in lieu of RT-PCR tests as part of COVID-19 public health response activities in Lima, Peru. To assess their utility, we explored the relationship between the time since onset of several COVID-19-related symptoms and the sensitivity of a rapid combined IgM/IgG antibody test., Methods: We collected data from a community sample of individuals (n = 492) who received concurrent RT-PCR and rapid IgM/IgG antibody testing between May 2020 and March 2021. We estimated the sensitivity of the antibody test, against the RT-PCR test, by weeks since symptom onset via segmented regression analysis., Results: The overall sensitivity of the rapid IgM/IgG antibody test was 46.7% (95% CI, 42.4-51.2%). Among 372 (75.6%) participants who reported COVID-19-related symptoms, sensitivity increased from 30.4% (95% CI, 24.7-36.6%) in week 1 after symptom onset to 83.3% (95% CI, 41.6-98.4%) in week 4. The test sensitivity increased by 31.9% (95% CI, 24.8-39.0%) per week until week 2 to 3, then decreased by - 6.0% (95% CI, - 25.7-13.7%) per week thereafter., Conclusion: Rapid antibody tests are a poor substitute for RT-PCR testing, regardless of presenting symptoms. This highlights the need for future pandemic planning to include timely and equitable access to gold-standard diagnostics, treatment, and vaccination., (© 2023. The Author(s).)

Serological antibody tests are useful complements of nuclei acid detection for SARS-CoV-2 diagnosis, which can significantly improve diagnostic accuracy. However, antibody detection in serum or plasma remains challenging to do with high sensitivity. In this study, Ag nanoparticles with ultra-thin Au shells embedded with 4-mercaptobenzoic acid (MBA) (Ag MBA @Au) were manufactured and then assembled onto Fe 3 O 4 surface by electrostatic interaction to construct the Fe 3 O 4 -Ag MBA @Au nanoparticles (NPs) with magnetic-Raman-colorimetric properties. Based on the composite nanoparticles, a colorimetric and Raman dual-mode lateral flow immunoassay (LFIA) for ultrasensitive identification of SARS-CoV-2 nucleocapsid (N) protein antibody was constructed. The magnetic nanoparticles (Fe 3 O 4 NPs) were acted as the core and coated a layer of Ag MBA @Au particles on the surface by electrostatic interaction to prepare Fe 3 O 4 -Ag MBA @Au NPs, which can amplify the SERS signal due to multiple Ag MBA @Au particles concentrated on a single magnetic nanoparticle. Moreover, the Fe 3 O 4 -Ag MBA @Au NPs facilitated pre-purifying sample using magnetic separation, and complex matrix interference would be greatly decreased in the detection. The Fe 3 O 4 -Ag MBA @Au NPs modified with N protein recognized and bound with N protein antibodies, which were trapped on the T-line, forming color band for observing detection. Under optimal conditions, the N protein antibodies could be qualitatively detected in colorimetric mode with the visual limit of 10 -8  mg/mL and quantitatively detected by SERS signals between 10 -6 and 10 -10  mg /mL with 0.08 pg/mL detection limit. The coefficients variations (CV) of intra-assay was 8.0%, whereas of inter-assay was 11.7%, confirming of good reproducibility. Finally, this approach was able to discriminate between positive, negative, and weakly positive samples when detecting 107 clinical serum samples. The process enables highly sensitive quantitative assays that are valuable for evaluating disease processes and guiding treatment. Colorimetric and Raman dual-mode LFIA detection of SARS-CoV-2 N protein antibody based on Fe 3 O 4 -Ag MBA @Au nanoparticles., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Vaccines are critical cost-effective tools to control the COVID-19 pandemic. The heterologous prime-boost vaccination has been used by many countries to overcome supply issues, so the effectiveness and safety of this strategy need to be better clarified. This study aims to verify the effect of heterologous prime-boost COVID-19 vaccination on healthcare professionals from Dante Pazzanese Hospital in Brazil. It was performed serological assays of vaccinated individuals after 2-dose of CoronaVac (Sinovac; n = 89) or ChAdOx1 nCoV-19 (Oxford-AstraZeneca; n = 166) followed by a BNT162b2 booster (Pfizer-BioNTech; n = 255). The serum antibodies anti-S (spike), anti-N (nucleocapsid), and anti-RBD (receptor binding domain) were assessed by enzyme-linked immunosorbent assay. The heterologous booster dose induced a 10-fold higher anti-Spike antibody regardless of the 2-dose of a prime vaccine. It was strikingly observed that BNT162b2 enhanced levels of anti-spike antibodies, even in those individuals who did not previously respond to the 2-dose of CoronaVac. In conclusion, the heterologous scheme of vaccination using mRNA as a booster vaccine efficiently enhanced the antibody response against SARS-CoV-2, especially benefiting those elderly who were seronegative with a virus-inactivated vaccine., (© 2022 Wiley Periodicals LLC.)

Infectious bursal disease (IBD) is an immunosuppressive and economically important disease of young chickens caused by infectious bursal disease virus (IBDV). The National Veterinary Institute (Bishoftu, Ethiopia) produces intermediate IBDV vaccine using primary chicken embryo fibroblast (CEF) cells, a method with technical and economical cumbersome. This study assessed the safety, immunogenicity, and efficacy of DF-1 cell line-adapted IBDV LC-75 vaccine strain in reference to the CEF-based vaccine. Confluent monolayer of DF-1 cells was infected with IBDV and cells with cytopathic effects were passaged until 3 rd passage. Viral growth was confirmed using a one-step RT-PCR targeting IBDV VP2 gene. Viral titer increased from 1 st passage through 3 rd passage. Safety was assessed in 30 specific-pathogen-free chickens (15 chickens/group) injected with 10-fold field dose of each vaccine intraocularly and monitored for 21 days. For immunogenicity and efficacy, 60 specific-pathogen-free chickens were grouped into 3 (20 chickens/group). First and 2 nd group received DF-1 cell and CEF-based IBDV vaccines, respectively. The 3 rd group served as unvaccinated control. Antibody response was measured using iELISA. Chickens were challenged 4 weeks postvaccination with very virulent IBDV (vvIBDV) intraocularly and followed-up for 10 days. Vaccination did not cause any adverse reactions during the 21 days of follow-up. In addition, both vaccines induced higher antibody titer 14 and 24 days-post-vaccination as compared to unvaccinated controls ( p < 0.05). Moreover, DF-1 and CEF-based IBDV LC-75 vaccines rendered a complete protection against vvIBDV. Contrarily, morbidity and mortality in unvaccinated chickens was 50% and 30%, respectively. The results indicated that DF-1 and CEF cell-based IBDV vaccines are comparably immunogenic and efficacious. Therefore, DF-1 cell-line can be considered an affordable and convenient alternative to the CEF-based approach. The suitability of DF-1 cells to grow other IBDV strains and safety of these vaccines on bursa of Fabricius should further be investigated., Competing Interests: The authors declare that they have no competing interests for this work., (Copyright © 2022 Daniel Workineh et al.)

Antibody detection assays are essential for evaluating immunity of individuals against a given virus, and this has been particularly relevant during the COVID-19 pandemic. Current serology assays either require a laboratory setting and take >1 hr (i.e., enzyme-linked immunosorbent assay [ELISA]) or are rapid but only qualitative in nature and cannot accurately track antibody levels over time (i.e., lateral flow assay [LFA]). Therefore, there is a need for development of a rapid and simple but also quantitative assay that can evaluate antibody levels in patients accurately over time. We have developed an assay that uses a split nanoluciferase fused to the spike or nucleocapsid proteins of the SARS-CoV-2 virus to enable luminescent-based detection of spike- or nucleocapsid-binding antibodies in serum, plasma, and whole blood samples. The resulting approach is simple, rapid, and quantitative and is highly amenable to low-/medium-throughput scale using plate-based assays, high-throughput scale using robotics, and point-of-care applications. In this article, we describe how to perform the assay in a laboratory setting using a plate reader or liquid-handling robotics and in a point-of-care setting using a handheld, battery-powered luminometer. Together, these assays allow antibody detection to be easily performed in multiple settings by simplifying and reducing assay time in a laboratory or clinical environment and by allowing for antibody detection in point-of-care, nonlaboratory settings. © 2022 Wiley Periodicals LLC. Basic Protocol: SARS-CoV-2 antibody detection using the split-luciferase assay on a medium-throughput scale with a laboratory luminometer Alternate Protocol 1: High-throughput-based protocol for SARS-CoV-2 antibody detection using a robotic platform Alternate Protocol 2: Point-of-care-based protocol for SARS-CoV-2 antibody detection using a handheld luminometer Support Protocol: Determining positive/negative cutoffs for test samples and standardizing the assay between days., (© 2022 Wiley Periodicals LLC.)

Surveillance is mandatory for tracking the progress of porcine reproductive and respiratory syndrome virus (PRRSV) control and elimination efforts in breeding herds. Processing fluids, the fluid recovered from tissues collected at castration and/or tail docking, are used for breeding herd surveillance by large segments of the industry, but the basic diagnostic characteristics of processing fluids are largely undescribed. We undertook 3 studies to address this information gap. In study 1, we found no differences among the PRRSV RT-rtPCR results obtained with 4 commercial RNA extraction kits. In study 2, we found that PRRSV RNA was highly stable in processing fluid samples at -20°C or 4°C, but detrimental effects were observed at ≥22°C within 24 h. In study 3, using a modified PRRSV ELISA at a sample:positive cutoff of ≥0.5, we found excellent discrimination in the detection of PRRSV antibody (IgM, IgA, IgG) in processing fluids from herds of known PRRSV status. Judicious handling of processing fluid samples from sow herds, and the use of methods available in veterinary diagnostic laboratories, can provide a foundation for reliable PRRSV surveillance.

Family oral fluids (FOFs) are an aggregate sample type shown to be a cost-efficient and convenient option for determining the porcine reproductive and respiratory syndrome virus (PRRSV) status of weaning age pigs. This study investigates the effect of pooling PRRSV-positive FOF samples with PRRSV-negative FOF samples at different levels (1/3, 1/5, 1/10, 1/20) on the probability of PRRSV RNA detection by reverse-transcription real-time polymerase chain reaction (RT-rtPCR). Mathematical models were built to assess how much the probability of RT-rtPCR PRRSV detection changed with increasing proportion of PRRSV-positive samples present within pools and how partially sampling a farrowing room influenced the probability of RT-rtPCR detection of PRRSV RNA in pooled samples at different prevalence scenarios. A general example of a guideline for FOF-based sampling under different prevalence scenarios to detect PRRSV RNA by RT-rtPCR with at least 95 % certainty is presented. At the sample level, the probability of detecting PRRSV RNA by RT-rtPCR decreased from 100 % to 87 %, 68 %, and 26 % when diluting up to 1/20 for PRRSV positive FOF having an initial Cycle threshold (Ct) below 34, between 34 and 36, or above 36, respectively. When PRRSV prevalence is near-zero (1 or 2 litters positive out of 56), the most cost-efficient farrowing room sampling strategy to detect PRRSV RNA with at least 95 % certainty was pooling FOF samples up to 1/10; at higher prevalence (≥ 3 of 56 litters positive), the most cost-efficient strategy was submitting samples in pools of 20. Subsampling a farrowing room for FOF pools was also demonstrated to be a valuable cost-saving strategy. Overall, based on the conditions of this study, pooling FOFs up to 1/20 is a valid option in situations of cost constraint and regardless of pooling level chosen, capturing as many litters as possible improves the probability of PRRSV detection., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Subacute Sclerosing Panencephalitis (SSPE) mainly affects children and young people. It is a rare, chronic progressive degenerative form of cerebral inflammation with various infectious noxa, which develops for years after a primary, uncomplicated infection, and the highest percentage can be caused by measles virus and in rare cases by rubella. The aim of the present study is to investigate in the laboratory the role of measles virus in the development of neurological symptoms and diseases of the CNS., Methods: A total of 46 clinical materials (23 sera samples and 23 CSF) obtained from 23 patients with neurological symptoms and diagnoses: "SSPE" (in 10 patients) and "Encephalitis" (in 13 patients), in the period January 2011 - December 2020 were tested in the National Reference Laboratory (NRL) "Measles, mumps and rubella" at National Centre of Infectious and Parasitic Diseases (NCIPD), Sofia, Bulgaria. Serological (indirect ELISA test for the detection of specific measles IgG/IgM antibodies in serum samples and cerebrospinal fluid) and molecular (RT-PCR for the demonstration of viral RNA) methods were used., Results: The study was performed by parallel testing of serum samples and CSF from each patient. Positive results for measles IgG antibodies in sera were found in 21 patients. Presence of measles IgG antibodies in CSF was demonstrated in four children with diagnosis SSPE (two children at 4 years, one child at 4 years and 6 months, and one at 11 years old). All children with positive laboratory results for SSPE had evidence of MeV infection before 2 years of age. The patients with SSPE had high antibody titers (CSF > 230 U/mL) in their CSF. Patients with positive anti-Measles IgG in the CSF were also found to have positive results for protective measles IgG in the serum samples and their IgG titers were nearly twice as high compared to other patients' sera. The presence of specific measles IgM antibodies was not demonstrated in the tested specimens. RT-PCR test was performed for all samples, and the presence of viral RNA was not detected., Conclusions: The measles infection can be a reason for developing serious complications affecting CNS in all age groups. SSPE itself is extremely difficult to diagnose, which is why laboratory confirmation of any clinical case is a necessary condition for effective disease surveillance.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been one of the major health-related concerns in the swine production industry. Through its rapid transmission and mutation, the simultaneous circulation of multiple PRRSV strains can be a challenge in PRRSV diagnostic, control and surveillance. The objective of this longitudinal study was to describe the temporal detection of PRRSV in swine farms with different production types and PRRS management strategies. Tonsil scraping (n = 344) samples were collected from three breeding and two growing herds for approximately one year. In addition, processing fluids (n = 216) were obtained from piglet processing batches within the three breeding farms while pen-based oral fluids (n = 125) were collected in the two growing pig farms. Viral RNA extraction and reverse-transcription quantitative PCR (RT-qPCR) were conducted for all samples. The sample positivity threshold was set at quantification cycle (Cq) of ≤ 37. Statistical analyses were performed using generalized linear modelling and post hoc pairwise comparisons with Bonferroni adjustments using R statistical software. The results suggested a higher probability of detection in processing fluids compared to tonsil scraping specimens [odds ratio (OR) = 3.86; p = .096] in breeding farms whereas oral fluids were outperformed by tonsil scrapings (OR = 0.26; p < .01) in growing pig farms. The results described herein may lead to an improvement in PRRSV diagnostic and surveillance by selecting proper specimens., (© 2021 Wiley-VCH GmbH.)

The Covid-19 pandemic, caused by SARS-CoV-2, has resulted in over 6 million reported deaths worldwide being one of the biggest challenges the world faces today. Here we present optimizations of all steps of an enzyme-linked immunosorbent assay (ELISA)-based test to detect IgG, IgA and IgM against the trimeric spike (S) protein, receptor binding domain (RBD), and N terminal domain of the nucleocapsid (N-NTD) protein of SARS-CoV-2. We discuss how to determine specific thresholds for antibody positivity and its limitations according to the antigen used. We applied the assay to a cohort of 126 individuals from Rio de Janeiro, Brazil, consisting of 23 PCR-positive individuals and 103 individuals without a confirmed diagnosis for SARS-CoV-2 infection. To illustrate the differences in serological responses to vaccinal immunization, we applied the test in 18 individuals from our cohort before and after receiving ChAdOx-1 nCoV-19 or CoronaVac vaccines. Taken together, our results show that the test can be customized at different stages depending on its application, enabling the user to analyze different cohorts, saving time, reagents, or samples. It is also a valuable tool for elucidating the immunological consequences of new viral strains and monitoring vaccination coverage and duration of response to different immunization regimens., (© 2022. The Author(s).)

Introduction: The COVID-19 Community Research Partnership is a population-based longitudinal syndromic and sero-surveillance study. The study includes over 17,000 participants from six healthcare systems in North Carolina who submitted over 49,000 serology results. The purpose of this study is to use these serology data to estimate the cumulative proportion of the North Carolina population that has either been infected with SARS-CoV-2 or developed a measurable humoral response to vaccination., Methods: Adult community residents were invited to participate in the study between April 2020 and February 2021. Demographic information was collected and daily symptom screen was completed using a secure, HIPAA-compliant, online portal. A portion of participants were mailed kits containing a lateral flow assay to be used in-home to test for presence of anti-SARS-CoV-2 IgM or IgG antibodies. The cumulative proportion of participants who tested positive at least once during the study was estimated. A standard Cox proportional hazards model was constructed to illustrate the probability of seroconversion over time up to December 20, 2020 (before vaccines available). A separate analysis was performed to describe the influence of vaccines through February 15, 2021., Results: 17,688 participants contributed at least one serology result. 68.7% of the population were female, and 72.2% were between 18 and 59 years of age. The average number of serology results submitted per participant was 3.0 (±1.9). By December 20, 2020, the overall probability of seropositivity in the CCRP population was 32.6%. By February 15, 2021 the probability among healthcare workers and non-healthcare workers was 83% and 49%, respectively. An inflection upward in the probability of seropositivity was demonstrated around the end of December, suggesting an influence of vaccinations, especially for healthcare workers. Among healthcare workers, those in the oldest age category (60+ years) were 38% less likely to have seroconverted by February 15, 2021., Conclusions: Results of this study suggest more North Carolina residents may have been infected with SARS-CoV-2 than the number of documented cases as determined by positive RNA or antigen tests. The influence of vaccinations on seropositivity among North Carolina residents is also demonstrated. Additional research is needed to fully characterize the impact of seropositivity on immunity and the ultimate course of the pandemic., Competing Interests: The authors have declared that no competing interests exist.

Background: In a previous cohort study of 4-year-old Danish children, revaccination with the live measles-mumps-rubella vaccine (MMR) was associated with a 16% reduction in the rate of hospitalization lasting two days or longer for non-measles-mumps-rubella infections., Aim: To examine if the introduction of revaccination with MMR at 4 years of age in Denmark (spring 2008) and at 7-9 years of age in Sweden (autumn 2009), at a time when there was virtually no measles, mumps or rubella cases, was associated with a reduction in the rate of hospitalization-for-infection lasting two days or longer at the population level., Methods: We included 4-year-olds in Denmark and 7-9-year-olds in Sweden. We obtained the number of hospitalization-for-infection lasting two days or longer from nationwide hospital registers. Person-years at risk were approximated from population statistics for each season and year. We performed an interrupted time series analysis using Poisson regression to estimate the change in hospitalization incidence rates following the introduction of MMR revaccination, adjusting for seasonality. We also performed analyses with control series (3-year-olds in Denmark and 4-year-olds in Sweden)., Results: Comparing the incidence of hospitalization-for-infection lasting two days or longer after the introduction of MMR revaccination with the expected level without an introduction of MMR revaccination resulted in an incidence rate ratio of 1.07 (95% confidence interval [CI] = 0.89-1.28) for 4-year-olds in Denmark and 0.89 (95% CI = 0.77-1.02) for 7-9-year-olds in Sweden in analyses without controls. Analyses with controls gave similar results., Conclusion: This population-level study of the introduction of MMR revaccination in Denmark and Sweden had inadequate power to confirm or refute the findings from an individual-level Danish study of an association between MMR revaccination and a lower incidence rate of hospitalization-for-infection lasting two days or longer., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: HN is an employee of the Finnish Institute for Health and Welfare, which has received research funding from GlaxoSmithKline, Sanofi-Pasteur and Pfizer for clinical trials. All other authors report no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: Synthesise evidence on production of SARS-CoV-2 antibodies in human milk of individuals who had COVID-19, and antibodies' ability to neutralise SARS-CoV-2 infectivity., Design: A systematic review of studies published from 1 December 2019 to 16 February 2021 without study design restrictions., Setting: Data were sourced from PubMed, MEDLINE, Embase, CNKI, CINAHL and WHO COVID-19 database. Search was also performed through reviewing references of selected articles, Google Scholar and preprint servers. Studies that tested human milk for antibodies to SARS-CoV-2 were included., Patients: Individuals with COVID-19 infection and human milk tested for anti-SARS-CoV-2 neutralising antibodies., Main Outcome Measures: The presence of neutralising antibodies in milk samples provided by individuals with COVID-19 infection., Results: Individual participant data from 161 persons (14 studies) were extracted and re-pooled. Milk from 133 (82.6%) individuals demonstrated the presence of anti-SARS-CoV-2 immunoglobulin A (IgA), IgM and/or IgG. Illness severity data were available in 146 individuals; 5 (3.4%) had severe disease, 128 (87.7%) had mild disease, while 13 (8.9%) were asymptomatic. Presence of neutralising antibodies in milk from 20 (41.7%) of 48 individuals neutralised SARS-CoV-2 infectivity in vitro. Neutralising capacity of antibodies was lost after Holder pasteurisation but preserved after high-pressure pasteurisation., Conclusion: Human milk of lactating individuals after COVID-19 infection contains anti-SARS-CoV-2-specific IgG, IgM and/or IgA, even after mild or asymptomatic infection. Current evidence demonstrates that these antibodies can neutralise SARS-CoV-2 virus in vitro. Holder pasteurisation deactivates SARS-CoV-2-specific IgA, while high-pressure pasteurisation preserves the SARS-CoV-2-specific IgA function., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Continuous emergence of SARS-CoV-2 variants of concern (VOCs) is fueling the COVID-19 pandemic. Omicron (B.1.1.529) rapidly spread worldwide. The large number of mutations in its Spike raise concerns about a major antigenic drift that could significantly decrease vaccine efficacy and infection-induced immunity. A long interval between BNT162b2 mRNA doses elicits antibodies that efficiently recognize Spikes from different VOCs. Here, we evaluate the recognition of Omicron Spike by plasma from a cohort of SARS-CoV-2 naive and previously infected individuals who received their BNT162b2 mRNA vaccine 16 weeks apart. Omicron Spike is recognized less efficiently than D614G, Alpha, Beta, Gamma, and Delta Spikes. We compare with plasma activity from participants receiving a short (4 weeks) interval regimen. Plasma from individuals of the long-interval cohort recognize and neutralize better the Omicron Spike compared with those who received a short interval. Whether this difference confers any clinical benefit against Omicron remains unknown., Competing Interests: Declaration of interests A.F. has filed a provisional patent application on the anti-Spike monoclonal antibody CV3-25., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objectives: To develop and validate a specific protocol for SARS-CoV-2 detection in breast milk matrix and to determine the impact of maternal SARS-CoV-2 infection on the presence, concentration and persistence of specific SARS-CoV-2 antibodies., Design and Patients: This is a prospective, multicentre longitudinal study (April-December 2020) in 60 mothers with SARS-CoV-2 infection and/or who have recovered from COVID-19. A control group of 13 women before the pandemic were also included., Setting: Seven health centres from different provinces in Spain., Main Outcome Measures: Presence of SARS-CoV-2 RNA in breast milk, targeting the N1 region of the nucleocapsid gene and the envelope (E) gene; presence and levels of SARS-CoV-2-specific immunoglobulins (Igs)-IgA, IgG and IgM-in breast milk samples from patients with COVID-19., Results: All breast milk samples showed negative results for presence of SARS-CoV-2 RNA. We observed high intraindividual and interindividual variability in the antibody response to the receptor-binding domain of the SARS-CoV-2 spike protein for each of the three isotypes IgA, IgM and IgG. Main Protease (MPro) domain antibodies were also detected in milk. 82.9% (58 of 70) of milk samples were positive for at least one of the three antibody isotypes, with 52.9% of these positive for all three Igs. Positivity rate for IgA was relatively stable over time (65.2%-87.5%), whereas it raised continuously for IgG (from 47.8% for the first 10 days to 87.5% from day 41 up to day 206 post-PCR confirmation)., Conclusions: Our study confirms the safety of breast feeding and highlights the relevance of virus-specific SARS-CoV-2 antibody transfer. This study provides crucial data to support official breastfeeding recommendations based on scientific evidence. Trial registration number NCT04768244., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Seroepidemiological studies to monitor antibody kinetics are important for assessing the extent and spread of SARS-CoV-2 in a population. Noninvasive sampling methods are advantageous for reducing the need for venipuncture, which may be a barrier to investigations, particularly in pediatric populations. Oral fluids are obtained by gingiva-crevicular sampling from children and adults and are very well accepted. Enzyme immunoassays (EIAs) based on these samples have acceptable sensitivity and specificity compared to conventional serum-based antibody EIAs and are suitable for population-based surveillance. We describe the development and evaluation of SARS-CoV-2 IgG EIAs using SARS-CoV-2 viral nucleoprotein (NP) and spike (S) proteins in IgG isotype capture format and an indirect receptor-binding-domain (RBD) IgG EIA, intended for use in children as a primary endpoint. All three assays were assessed using a panel of 1,999 paired serum and oral fluids from children and adults participating in school SARS-CoV-2 surveillance studies during and after the first and second pandemic wave in the United Kingdom. The anti-NP IgG capture assay was the best candidate, with an overall sensitivity of 75% (95% confidence interval [CI]: 71 to 79%) and specificity of 99% (95% CI: 78 to 99%) compared with paired serum antibodies. Sensitivity observed in children (80%, 95% CI: 71 to 88%) was higher than that in adults (67%, CI: 60% to 74%). Oral fluid assays (OF) using spike protein and RBD antigens were also 99% specific and achieved reasonable but lower sensitivity in the target population (78%, 95% CI [68% to 86%] and 53%, 95% CI [43% to 64%], respectively). IMPORTANCE We report on the first large-scale assessment of the suitability of oral fluids for detection of SARS-CoV-2 antibody obtained from healthy children attending school. The sample type (gingiva-crevicular fluid, which is a transudate of blood but is not saliva) can be self collected. Although detection of antibodies in oral fluids is less sensitive than that in blood, our study suggests an optimal format for operational use. The laboratory methods we have developed can reliably measure antibodies in children, who are able to take their own samples. Our findings are of immediate practical relevance for use in large-scale seroprevalence studies designed to measure exposure to infection, as they typically require venipuncture. Overall, our data indicate that OF assays based on the detection of SARS-CoV-2 antibodies are a tool suitable for population-based seroepidemiology studies in children and highly acceptable in children and adults, as venipuncture is no longer necessary.

Background: Following the full re-opening of schools in England and emergence of the SARS-CoV-2 Alpha variant, we investigated the risk of SARS-CoV-2 infection in students and staff who were contacts of a confirmed case in a school bubble (school groupings with limited interactions), along with their household members., Methods: Primary and secondary school bubbles were recruited into sKIDsBUBBLE after being sent home to self-isolate following a confirmed case of COVID-19 in the bubble. Bubble participants and their household members were sent home-testing kits comprising nasal swabs for RT-PCR testing and whole genome sequencing, and oral fluid swabs for SARS-CoV-2 antibodies., Results: During November-December 2020, 14 bubbles were recruited from 7 schools, including 269 bubble contacts (248 students, 21 staff) and 823 household contacts (524 adults, 299 children). The secondary attack rate was 10.0% (6/60) in primary and 3.9% (4/102) in secondary school students, compared to 6.3% (1/16) and 0% (0/1) among staff, respectively. The incidence rate for household contacts of primary school students was 6.6% (12/183) and 3.7% (1/27) for household contacts of primary school staff. In secondary schools, this was 3.5% (11/317) and 0% (0/1), respectively. Household contacts were more likely to test positive if their bubble contact tested positive although there were new infections among household contacts of uninfected bubble contacts., Interpretation: Compared to other institutional settings, the overall risk of secondary infection in school bubbles and their household contacts was low. Our findings are important for developing evidence-based infection prevention guidelines for educational settings., Competing Interests: The authors have declared that no competing interests exist.

Background: Immunoglobulin A (IgA) is an important component of the early immune response to SARS-CoV-2. Prior serosurveys in high-risk groups employing IgG testing alone have provided discordant estimates. The potential added benefit of IgA in serosurveys has not been established., Methods: Longitudinal serosurvey of first responders (police, emergency medical service providers, fire fighters, and other staff) employing 3 serologic tests (anti-spike IgA, anti-spike IgG, and anti-nucleocapsid IgG) correlated with surveys assessing occupational and nonoccupational risk, exposure to COVID-19, and illnesses consistent with COVID-19., Results: Twelve percent of first responders in Colorado at baseline and 22% at follow-up were assessed as having SARS-CoV-2 infection. Five percent at baseline and 6% at follow-up were seropositive only for IgA. Among those IgA positive only at baseline, the majority (69%) had a positive antibody at follow-up; 45% of those infected at baseline and 33% at follow-up were asymptomatic. At all time points, the estimated cumulative incidence in our study was higher than that in the general population., Conclusions: First responders are at high risk of infection with SARS-CoV-2. IgA testing identified a significant portion of cases missed by IgG testing and its use as part of serologic surveys may improve retrospective identification of asymptomatic infection., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Antibodies in milk obtained from those previously SARS-CoV-2-infected or vaccinated against COVID-19 may provide passive immunity to the breastfed infant. Few assays have been established to measure antibodies in human milk, despite the public health importance of this topic. In the present protocol, we describe an optimized indirect ELISA assay aimed to measure SARS-CoV-2-reactive antibodies in human milk, which can be used as a rapid screen on undiluted samples or to designate samples as relatively low, moderate, or high titer. For complete details on the use and execution of this protocol, please refer to Fox et al. (2020)., Competing Interests: Mount Sinai has filed for patent protection for this assay., (© 2022 The Author(s).)

Mucosal immunity plays a crucial role in controlling upper respiratory infections, including influenza. We established a quantitative ELISA to measure the amount of influenza virus-specific salivery IgA (sIgA) and salivary IgG (sIgG) antibodies using a standard antibody broadly reactive to the influenza A virus. We then analyzed saliva and serum samples from seven individuals infected with the A(H1N1)pdm09 influenza virus during the 2019-2020 flu seasons. We detected an early (6-10 days post-infection) increase of sIgA in five of the seven samples and a later (3-5 weeks) increase of sIgG in six of the seven saliva samples. Although the conventional parenteral influenza vaccine did not induce IgA production in saliva, vaccinated individuals with a history of influenza infection had higher basal levels of sIgA than those without a history. Interestingly, we observed sIgA and sIgG in an asymptomatic individual who had close contact with two influenza cases. Both early mucosal sIgA secretion and late systemically induced sIgG in the mucosal surface may protect against virus infection. Despite the small sample size, our results indicate that the saliva test system can be useful for analyzing upper mucosal immunity in influenza., Competing Interests: The authors have declared that no competing interests exist.