The Luciferase Immunoprecipitation System (LIPS) Targeting the Spike Protein of SARS-CoV-2 Is More Accurate than Nucleoprotein-Based LIPS and ELISAs for Mink Serology

Since anthropo-zoonotic outbreaks of SARS-CoV-2 have been reported in mink farms, it is important to monitor the seroprevalence within this population. To investigate the accuracy of nucleo (N) or spike (S) protein-based assays to detect anti-SARS-CoV-2 antibodies in animal serum, we compared four assays, two commercial N-based enzyme-linked immunosorbent assays (ELISA) validated for animal sera and two luciferase immunoprecipitation systems (LIPS-N and LIPS-S), to the reference standard plaque reduction neutralisation test (PRNT). Samples included in this study were derived from a naturally infected mink population. For the first time in this study, serum samples of mink were collected over a 307-day period, at different time points, thus providing an overview of performances of four different rapid serological tests over time. The assays were compared by performing a correlation analysis using R2, Spearman's rank-order correlation coefficient, and Fleiss' and Cohen's kappa for analysis of agreement to PRNT, and an UpSet chart was created to visualize the number of shared positive samples between assays. Cohen's kappa test on categorical data showed an excellent agreement between PRNT and LIPS-S, while agreements between PRNT and N-based methods decreased from fair for LIPS-N to poor agreements for the ELISA kits. In addition, LIPS-S revealed the highest number of true-positive SARS-CoV-2 samples compared to N-based methods. Despite an excellent agreement between LIPS-S and PRNT, a weak correlation was detectable between PRNT titres and relative light units. This study shows that the LIPS-S assay can be used for serological surveillance within a naturally exposed mink population, while N-based serological assays are less accurate providing a higher number of false-negative results, especially at a later stage of infection, thus indicating that N antibodies are less persistent in naturally exposed mink. Our findings provide crucial information for veterinarians and competent authorities involved in surveillance and outbreak investigation in wild and farmed minks. [ FROM AUTHOR] Copyright of Transboundary & Emerging Diseases is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Long-term Immune Response to SARS-CoV-2 Infection Among Children and Adults After Mild Infection.

Importance: Understanding the long-term immune response against SARS-CoV-2 infection in children is crucial to optimize vaccination strategies. Although it is known that SARS-CoV-2 antibodies may persist in adults 12 months after infection, data are limited in the pediatric population. Objective: To examine long-term anti-SARS-CoV-2 spike receptor-binding domain (S-RBD) IgG kinetics in children after SARS-CoV-2 infection. Design, Setting, and Participants: In this single-center, prospective cohort study, patients were enrolled consecutively from April 1, 2020, to August 31, 2021, at the COVID-19 Family Cluster Follow-up Clinic, Department of Women's and Children's Health, University Hospital of Padua. A cohort of 252 COVID-19 family clusters underwent serologic follow-up at 1 to 4, 5 to 10, and more than 10 months after infection with quantification of anti-S-RBD IgG by chemiluminescent immunoassay. Exposures: SARS-CoV-2 infection. Results: Among 902 study participants, 697 had confirmed SARS-CoV-2 infection, including 351 children or older siblings (mean [SD] age, 8.6 [5.1] years) and 346 parents (mean [SD] age, 42.5 [7.1] years). Among 697 cases, 674 (96.7%) were asymptomatic or mild. Children had significantly higher S-RBD IgG titers than older patients across all follow-up time points, with an overall median S-RBD IgG titer in patients younger than 3 years 5-fold higher than adults (304.8 [IQR, 139.0-516.6] kBAU/L vs 55.6 [24.2-136.0] kBAU/L, P < .001). Longitudinal analysis of 56 study participants sampled at least twice during follow-up demonstrated the persistence of antibodies up to 10 months from infection in all age classes, despite a progressive decline over time. Conclusions and Relevance: In this cohort study of Italian children and adults following SARS-CoV-2 infection different kinetics of SARS-CoV-2 antibodies were found across several age classes of individuals with asymptomatic or mild COVID-19, which could help in optimizing COVID-19 vaccination strategies and prevention policies. This work provides further evidence of sustained immune response in children up to 1 year after primary SARS-CoV-2 infection.

Pulmonary fibrosis in a dog as a sequela of infection with Severe Acute Respiratory Syndrome Coronavirus 2? A case report.

BACKGROUND: Interstitial lung disease is a heterogeneous group of conditions characterized by severe radiographic changes and clinicopathological findings. However, in the vast majority of cases, the cause remains unknown. CASE DESCRIPTION: In the present study, we reported the clinical case of a 3 years old female Bull Terrier presented in October 2020 to the Advanced Diagnostic Imaging Department of the Turin Veterinary Teaching Hospital with a progressive pulmonary illness characterized by dyspnea, exercise intolerance, and a diffuse and severe pulmonary interstitial pattern at imaging investigations. Considering the clinical findings, the dog was included in a serological survey for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection in companion animals, showing positive results. Due to the further clinical worsening, the owners opted for euthanasia. At necroscopy, dog showed severe and chronic bronchopneumonia compatible with a Canine Idiopathic Pulmonary Fibrosis and with serological features linked to a SARS-CoV-2 infection. CONCLUSIONS: The comparison of these lesions with those reported in humans affected by Coronavirus Disease 2019 (COVID-19) supports the hypothesis that these findings may be attributable to the post-acute sequelae of SARS-CoV-2 infection in a dog with breed predisposition to Canine Idiopathic Pulmonary Fibrosis (CIPF), although direct evidence of SARS-CoV-2 by molecular or antigenic approaches remained unsolved.

Detailed epitope mapping of SARS-CoV-2 nucleoprotein reveals specific immunoresponse in cats and dogs housed with COVID-19 patients.

Since the initial emergence in December 2019, the novel Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been reported in over 200 countries, representing an unprecedented challenge related to disease control worldwide. In this context, cases of human to animal transmission have been reported, raising concern about the potential role of companion animals in the pandemic and stressing the need for reliable animal testing. In the study, a detailed epitope mapping of SARS-CoV-2 nucleoprotein, using both human and pet sera, allowed the identification of the most antigenic region in the C-terminus domain of the protein, which was used to develop an experimental double antigen-based ELISA. A panel of pre-pandemic sera and sera of animals immunized against (or naturally infected with) related coronaviruses was used to assess assay specificity at 99.5%. Positive sera belonging to animals housed with COVID-19 patients were confirmed with the experimental double-antigen ELISA using Plaque Reduction Neutralization test (PRNT) test as gold standard. The availability of a serological assay that targets a highly specific viral antigen represents a valuable tool for multispecies monitoring of Coronavirus Disease 2019 (COVID-19) infection in susceptible animals.

Neutralizing antibody titers six months after Comirnaty vaccination: kinetics and comparison with SARS-CoV-2 immunoassays.

OBJECTIVES: mRNA vaccines, including Comirnaty (BNT162b2 mRNA, BioNTech-Pfizer), elicit high IgG and neutralizing antibody (NAb) responses after the second dose, but the progressive decrease in serum antibodies against SARS-CoV-2 following vaccination have raised questions concerning long-term immunity, decreased antibody levels being associated with breakthrough infections after vaccination, prompting the consideration of booster doses. METHODS: A total number of 189 Padua University-Hospital healthcare workers (HCW) who had received a second vaccine dose were asked to collect serum samples for determining Ab at 12 (t12) and 28 (t28) days, and 6 months (t6m) after their first Comirnaty/BNT162b2 inoculation. Ab titers were measured with plaque reduction neutralization test (PRNT), and three chemiluminescent immunoassays, targeting the receptor binding domain (RBD), the trimeric Spike protein (trimeric-S), and surrogate viral neutralization tests (sVNT). RESULTS: The median percentages (interquartile range) for decrease in antibodies values 6 months after the first dose were 86.8% (67.1-92.8%) for S-RBD IgG, 82% (58.6-89.3%) for trimeric-S, 70.4% (34.5-86.4%) for VNT-Nab, 75% (50-87.5%) for PRNT50 and 75% (50-93.7%) for PRNT90. At 6 months, neither PRNT titers nor VNT-Nab and S-RBD IgG bAb levels correlated with age (p=0.078) or gender (p=0.938), while they were correlated with previous infection (p<0.001). CONCLUSIONS: After 6 months, a method-independent reduction of around 90% in anti-SARS-CoV-2 antibodies was detected, while no significant differences were found between values of males and females aged between 24 and 65 years without compromised health status. Further efforts to improve analytical harmonization and standardization are needed.

SARS-CoV-2 neutralizing antibodies after one or two doses of Comirnaty (BNT162b2, BioNTech/Pfizer): Kinetics and comparison with chemiluminescent assays.

BACKGROUND: Studies evaluating neutralizing antibody (NAb) after BNT162b2 vaccine are scarce. We therefore compared NAb using the plaque reduction neutralization test (PRNT) in vaccinated subjects, with those from five chemiluminescent (CLIA) assays, two targeting ACE and S-RBD interaction. METHODS: Sera from 174 completely Comirnaty/BNT162b2 vaccinated healthcare workers (HCW) were evaluated at t12 and t28. NAb titers at low (PRNT50) or high (PRNT90) stringency were compared with: Liaison SARS-CoV-2 Trimeric-S IgG, Elecsys S-RBD Ab, Maglumi SARS-CoV-2 S-RBD IgG and SARS-CoV-2 Nab; iFlash 2019-nCoV NAb. RESULTS: Neither PRNT50 nor PRNT90 correlated with age (range, 24-65 years); no significant differences were found for gender. PRNT50 and PRNT90 seropositive titers (≥1:20) were 43 (24.7%) and 15 (8.6%) at t12 and 167 (95.9%) and 149 (85.6%) at t28. CLIA results at t28 were uncorrelated with age, apart from Elecsys S-RBD Ab (r = -0.164, p = 0.046). Gender differences were found for Maglumi SARS-CoV-2 S-RBD IgG (p = 0.037) and Maglumi NAb (p = 0.046). Considering PRNT50 at thresholds of 1:20 (or 1:40) and 1:160 (or 1:320), corresponding to different immune protective levels, CLIA cut-offs have been identified. CONCLUSIONS: Comirnaty/BNT162b2 elicits strong NAb production, especially 28 days after first inoculum. Differences in correlation between Nab titers and circulating antibodies measured by 5 immunoassays have been found, being stronger the correlation for Maglumi Nab.

SARS-CoV-2 infection and replication in human gastric organoids.

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.

Potential of an Eco-Sustainable Probiotic-Cleaning Formulation in Reducing Infectivity of Enveloped Viruses.

The COVID-19 pandemic has deeply influenced sanitization procedures, and high-level disinfection has been massively used to prevent SARS-CoV-2 spread, with potential negative impact on the environment and on the threat of antimicrobial resistance (AMR). Aiming to overcome these concerns, yet preserving the effectiveness of sanitization against enveloped viruses, we assessed the antiviral properties of the Probiotic Cleaning Hygiene System (PCHS), an eco-sustainable probiotic-based detergent previously proven to stably abate pathogen contamination and AMR. PCHS (diluted 1:10, 1:50 and 1:100) was tested in comparison to common disinfectants (70% ethanol and 0.5% sodium hypochlorite), in suspension and carrier tests, according with the European UNI EN 14476:2019 and UNI EN 16777:2019 standards. Human alpha- and beta-coronaviruses hCoV-229E and SARS-CoV-2, human herpesvirus type 1, human and animal influenza viruses, and vaccinia virus were included in the study. The results showed that PCHS was able to inactivate 99.99% of all tested viruses within 1-2 h of contact, both in suspension and on surface. Notably, while control disinfectants became inactive within 2 h after application, the PCHS antiviral action persisted up to 24 h post-application, suggesting that its use may effectively allow a continuous prevention of virus spread via contaminated environment, without worsening environmental pollution and AMR concern.

Asymptomatic and Mild SARS-CoV-2 Infections Elicit Lower Immune Activation and Higher Specific Neutralizing Antibodies in Children Than in Adults.

Background: The immune response plays a pivotal role in dictating the clinical outcome in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected adults, but it is still poorly investigated in the pediatric population. Methods: Of 209 enrolled subjects, 155 patients were confirmed by PCR and/or serology as having coronavirus disease 2019 (COVID-19). Blood samples were obtained at a median of 2.8 (interquartile, 2.1-3.7) and 6.1 (5.3-7.2) months after baseline (symptom onset and/or first positive virus detection). The immune profiles of activation, senescence, exhaustion, and regulatory cells were analyzed by flow cytometry. Neutralizing antibodies (nAbs) were detected by a plaque reduction neutralization test. In available nasopharyngeal swabs at baseline, SARS-CoV-2 levels were quantified by digital droplet PCR (ddPCR). Results: Overall, COVID-19 patients had higher levels of immune activation, exhaustion, and regulatory cells compared to non-COVID-19 subjects. Within the COVID-19 group, activated and senescent cells were higher in adults than in children and inversely correlated with the nAbs levels. Conversely, Tregs and Bregs regulatory cells were higher in COVID-19 children compared to adults and positively correlated with nAbs. Higher immune activation still persisted in adults after 6 months of infection, while children maintained higher levels of regulatory cells. SARS-CoV-2 levels did not differ among age classes. Conclusions: Adults displayed higher immune activation and lower production of anti-SARS-CoV-2 nAbs than children. The different immune response was not related to different viral load. The higher expression of regulatory cells in children may contribute to reduce the immune activation, thus leading to a greater specific response against the virus.

Serological Detection of SARS-CoV-2 Antibodies in Naturally-Infected Mink and Other Experimentally-Infected Animals.

The recent emergence of SARS-CoV-2 in humans from a yet unidentified animal reservoir and the capacity of the virus to naturally infect pets, farmed animals and potentially wild animals has highlighted the need for serological surveillance tools. In this study, the luciferase immunoprecipitation systems (LIPS), employing the spike (S) and nucleocapsid proteins (N) of SARS-CoV-2, was used to examine the suitability of the assay for antibody detection in different animal species. Sera from SARS-CoV-2 naturally-infected mink (n = 77), SARS-CoV-2 experimentally-infected ferrets, fruit bats and hamsters and a rabbit vaccinated with a purified spike protein were examined for antibodies using the SARS-CoV-2 N and/or S proteins. From comparison with the known neutralization status of the serum samples, statistical analyses including calculation of the Spearman rank-order-correlation coefficient and Cohen's kappa agreement were used to interpret the antibody results and diagnostic performance. The LIPS immunoassay robustly detected the presence of viral antibodies in naturally infected SARS-CoV-2 mink, experimentally infected ferrets, fruit bats and hamsters as well as in an immunized rabbit. For the SARS-CoV-2-LIPS-S assay, there was a good level of discrimination between the positive and negative samples for each of the five species tested with 100% agreement with the virus neutralization results. In contrast, the SARS-CoV-2-LIPS-N assay did not consistently differentiate between SARS-CoV-2 positive and negative sera. This study demonstrates the suitability of the SARS-CoV-2-LIPS-S assay for the sero-surveillance of SARS-CoV-2 infection in a range of animal species.

Impact of Full Vaccination with mRNA BNT162b2 on SARS-CoV-2 Infection: Genomic and Subgenomic Viral RNAs Detection in Nasopharyngeal Swab and Saliva of Health Care Workers.

SARS-CoV-2 infection was monitored in 1898 health care workers (HCWs) after receiving full vaccination with BNT162b2. Untill 30 June 2021, 10 HCWs tested positive for SARS-CoV-2 using real time RT-PCR, resulting in a 4-month cumulative incidence of 0.005%. The infection was mildly symptomatic in six (60%) and asymptomatic in four (40%) individuals. Among the infected HCWs, eight consenting individuals provided paired NPS and saliva during the course of infection, for the purpose of the analysis performed in the present study. Genomic and subgenomic viral RNAs were investigated using real-time RT-PCR in both biological specimens. The temporal profile of viral load was measured using ddPCR. Viral mutations were also analysed. Subgenomic viral RNA was detected in 8/8 (100%) NPS and in 6/8 (75%) saliva specimens at the baseline. The expression of subgenomic RNA was observed for up to 7 days in 3/8 (38%) symptomatic cases. Moreover, concordance was observed between NPS and saliva in the detection of viral mutations, and both N501Y and 69/70del (associated with the B.1.1.7 variant) were detected in the majority 6/8 (75%) of subjects, while the K417T mutation (associated with the P.1-type variants) was detected in 2/8 (25%) individuals. Overall, our findings report a low frequency of infected HCWs after full vaccination. It is, therefore, important to monitor the vaccinees in order to identify asymptomatic infected individuals. Saliva can be a surrogate for SARS-CoV-2 surveillance, particularly in social settings such as hospitals.

Prevalence of SARS-CoV-2 RNA on inanimate surfaces: a systematic review and meta-analysis.

Coronavirus disease (COVID-19) is a respiratory disease affecting many people and able to be transmitted through direct and perhaps indirect contact. Direct contact transmission, mediated by aerosols or droplets, is widely demonstrated, whereas indirect transmission is only supported by collateral evidence such as virus persistence on inanimate surfaces and data from other similar viruses. The present systematic review aims to estimate SARS-CoV-2 prevalence on inanimate surfaces, identifying risk levels according to surface characteristics. Data were obtained from studies in published papers collected from two databases (PubMed and Embase) with the last search on 1 September 2020. Included studies had to be papers in English, had to deal with coronavirus and had to consider inanimate surfaces in real settings. Studies were coded according to our assessment of the risk that the investigated surfaces could be contaminated by SARS-CoV-2. A meta-analysis and a metaregression were carried out to quantify virus RNA prevalence and to identify important factors driving differences among studies. Thirty-nine out of forty retrieved paper reported studies carried out in healthcare settings on the prevalence of virus RNA, five studies carry out also analyses through cell culture and six tested the viability of isolated viruses. Overall prevalences of SARS-CoV-2 RNA on high-, medium- and low-risk surfaces were 0.22 (CI95 [0.152-0.296]), 0.04 (CI95 [0.007-0.090]), and 0.00 (CI95 [0.00-0.019]), respectively. The duration surfaces were exposed to virus sources (patients) was the main factor explaining differences in prevalence.

Left ventricular longitudinal strain alterations in asymptomatic or mildly symptomatic paediatric patients with SARS-CoV-2 infection.

AIMS: Compared with adult patients, clinical manifestations of children's coronavirus disease-2019 (COVID-19) are generally perceived as less severe. The objective of this study was to evaluate cardiac involvement in previously healthy children with asymptomatic or mildly symptomatic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. METHODS AND RESULTS: We analysed a cohort of 53 paediatric patients (29 males, 55%), mean age 7.5 ± 4.7 years, who had a confirmed diagnosis of SARS-CoV-2 infection and were asymptomatic or only mildly symptomatic for COVID-19. Patients underwent standard transthoracic echocardiogram and speckle tracking echocardiographic study at least 3 months after diagnosis. Thirty-two age, sex, and body surface area comparable healthy subjects were used as control group. Left ventricular ejection fraction was within normal limits but significantly lower in the cases group compared to controls (62.4 ± 4.1% vs. 65.2 ± 5.5%; P = 0.012). Tricuspid annular plane systolic excursion (20.1 ± 3 mm vs. 19.8 ± 3.4 mm; P = 0.822) and left ventricular (LV) global longitudinal strain (-21.9 ± 2.4% vs. -22.6 ± 2.5%; P = 0.208) were comparable between the two groups. Regional LV strain analysis showed a significant reduction of the LV mid-wall segments strain among cases compared to controls. Furthermore, in the cases group, there were 14 subjects (26%) with a regional peak systolic strain below -16% (-2.5 Z score in our healthy cohort) in at least two segments. These subjects did not show any difference regarding symptoms or serological findings. CONCLUSION: SARS-CoV-2 infection may affect left ventricular deformation in 26% of children despite an asymptomatic or only mildly symptomatic acute illness. A follow-up is needed to verify the reversibility of these alterations and their impact on long-term outcomes.

Virological and immunological features of SARS-COV-2 infected children with distinct symptomatology.

BACKGROUND: Although SARS-CoV-2 immunizations have started in most countries, children are not currently included in the vaccination programs; thus, it remains crucial to define their anti-SARS-CoV-2 immune response in order to minimize the risk for other epidemic waves. This study sought to provide a description of the virology ad anti-SARS-CoV-2 immunity in children with distinct symptomatology. METHODS: Between March and July 2020, we recruited 15 SARS-CoV-2 asymptomatic (AS) and 51 symptomatic (SY) children, stratified according to WHO clinical classification. We measured SARS-CoV-2 viral load using ddPCR and qPCR in longitudinally collected nasopharyngeal swab samples. To define anti-SARS-CoV-2 antibodies, we measured neutralization activity and total IgG load (DiaSorin). We also evaluated antigen-specific B and CD8+T cells, using a labeled S1+S2 protein and ICAM expression, respectively. Plasma protein profiling was performed with Olink. RESULTS: Virological profiling showed that AS patients had lower viral load at diagnosis (p = .004) and faster virus clearance (p = .0002) compared with SY patients. Anti-SARS-CoV-2 humoral and cellular response did not appear to be associated with the presence of symptoms. AS and SY patients showed similar titers of SARS-CoV-2 IgG, levels of neutralizing activity, and frequency of Ag-specific B and CD8+ T cells, whereas pro-inflammatory plasma protein profile was found to be associated with symptomatology. CONCLUSION: We demonstrated the development of anti-SARS-CoV-2 humoral and cellular response with any regard to symptomatology, suggesting the ability of both SY and AS patients to contribute toward herd immunity. The virological profiling of AS patients suggested that they have lower virus load associated with faster virus clearance.

Mild SARS-CoV-2 Infections and Neutralizing Antibody Titers.

BACKGROUND: Recent evidence suggests that neutralizing antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 may persist over time; however, knowledge regarding pediatric subjects is limited. METHODS: A single-center, prospective observational study was conducted on 57 family clusters of coronavirus disease 2019, including children of neonatal and pediatric age attending the University Hospital of Padua (Italy). For each patient, blood samples were collected for both the quantification of nAbs through a plaque reduction neutralizing test and the detection of antinucleocapsid-spike protein immunoglobulin G and/or immunoglobulin M. RESULTS: We analyzed 283 blood samples collected from 152 confirmed coronavirus disease 2019 cases (82 parents and 70 children or older siblings of median age of 8 years, interquartile range: 4-13), presenting asymptomatic or with mildly symptomatic disease. Despite the decrease of immunoglobulin G over time, nAbs were found to persist up to 7 to 8 months in children, whereas adults recorded a modest declining trend. Interestingly, children aged <6 years, and, in particular, those aged <3 years, developed higher long-lasting levels of nAbs compared with older siblings and/or adults. CONCLUSIONS: Mild and asymptomatic severe acute respiratory syndrome coronavirus 2 infections in family clusters elicited higher nAbs among children.

SARS-Cov-2 Natural Infection in a Symptomatic Cat: Diagnostic, Clinical and Medical Management in a One Health Vision.

Despite the reported increase in SARS-CoV-2-infected pets, the description of the clinical features from natural infection and the medical follow up in symptomatic pets is still not sufficiently documented. This study reports the case of an indoor cat that displayed respiratory signs and a gastrointestinal syndrome, following the COVID-19 diagnosis of his owners. Thoracic radiographies were suggestive of bronchial pneumonia, while blood tests were indicative of a mild inflammatory process. Nasal and oropharyngeal swabs tested positive through RT-qPCR assays targeting SARS-CoV-2 genes 14 days after his owners tested positive for the virus. Nasal swabs persisted to be RT-qPCR positive after 31 days. Serology confirmed the presence of antibodies through ELISA, electrochemiluminescence analysis and plaque reduction neutralization test, recording a high antibody titre after 31 days. The cat improved after medical treatment and clinically recovered. This study suggests that exposure to SARS-CoV-2 could lead to a natural infection with bronchial pneumonia in cats along with a possible prolonged persistence of SARS-CoV-2 RNA in the upper airways, albeit at a low level. The cat developed neutralizing antibodies, reaching a high titre after 31 days. Further descriptions of SARS-CoV-2 naturally infected pets, their medical management and diagnostic findings would be useful to enhance knowledge about COVID-19 in susceptible animals.

Analytical and clinical performances of a SARS-CoV-2 S-RBD IgG assay: comparison with neutralization titers.

OBJECTIVES: SARS-CoV-2 serology presents an important role in several aspects of COVID-19 pandemic. Immunoassays performances have to be accurately evaluated and correlated with neutralizing antibodies. We investigated the analytical and clinical performances of a SARS-CoV-2 RBD IgG assay, automated on a high throughput platform, and the correlation of the antibodies (Ab) levels with the plaque reduction neutralization (PRNT50) Ab titers. METHODS: A series of 546 samples were evaluated by SARS-CoV-2 RBD IgG assay (Snibe diagnostics), including 171 negative and 168 positive SARS-CoV-2 subjects and a further group of 207 subjects of the COVID-19 family clusters follow-up cohort. RESULTS: Assay imprecision ranged from 3.98 to 12.18% being satisfactory at low and medium levels; linearity was excellent in all the measurement range. Considering specimens collected after 14 days post symptoms onset, overall sensitivity and specificity were 99.0 and 92.5%, respectively. A total of 281 leftover samples results of the PRNT50 test were available. An elevated correlation was obtained between the SARS-CoV-2 RBD IgG assay and the PRNT50 titer at univariate (ρ=0.689) and multivariate (ρ=0.712) analyses. CONCLUSIONS: SARS-CoV-2 S-RBD IgG assay shows satisfactory analytical and clinical performances, and a strong correlation with sera neutralizing activity.

Virological and immunological features of SARS-CoV-2-infected children who develop neutralizing antibodies.

As the global COVID-19 pandemic progresses, it is paramount to gain knowledge on adaptive immunity to SARS-CoV-2 in children to define immune correlates of protection upon immunization or infection. We analyzed anti-SARS-CoV-2 antibodies and their neutralizing activity (PRNT) in 66 COVID-19-infected children at 7 (±2) days after symptom onset. Individuals with specific humoral responses presented faster virus clearance and lower viral load associated with a reduced in vitro infectivity. We demonstrated that the frequencies of SARS-CoV-2-specific CD4+CD40L+ T cells and Spike-specific B cells were associated with the anti-SARS-CoV-2 antibodies and the magnitude of neutralizing activity. The plasma proteome confirmed the association between cellular and humoral SARS-CoV-2 immunity, and PRNT+ patients show higher viral signal transduction molecules (SLAMF1, CD244, CLEC4G). This work sheds lights on cellular and humoral anti-SARS-CoV-2 responses in children, which may drive future vaccination trial endpoints and quarantine measures policies.

A systematic review of human coronaviruses survival on environmental surfaces.

The current pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led people to implement preventive measures, including surface disinfection and use of alcohol-based hand gel, in order to avoid viral transmission via fomites. However, the role of surface transmission is still debated. The present systematic review aims to summarize all the evidence on surface survival of coronaviruses infecting humans. The analysis of 18 studies showed the longest coronavirus survival time is 28 days at room temperature (RT) on different surfaces: polymer banknotes, vinyl, steel, glass, and paper banknotes. Concerning SARS-CoV-2 human infection from contaminated surfaces, dangerous viral load on surfaces for up to 21 days was determined on polymer banknotes, steel, glass and paper banknotes. For viruses other than SARS-CoV-2, the longest period of survival was 14 days, recorded on glass. Environmental conditions can affect virus survival, and indeed, low temperatures and low humidity support prolonged survival of viruses on contaminated surfaces independently of surface type. Furthermore, it has been shown that exposure to sunlight significantly reduces the risk of surface transmission. Although studies are increasingly investigating the topic of coronavirus survival, it is difficult to compare them, given the methodology differences. For this reason, it is advisable to define a reference working protocol for virus survival trials, but, as an immediate measure, there is also a need for further investigations of coronavirus survival on surfaces.

Analytical and clinical performances of five immunoassays for the detection of SARS-CoV-2 antibodies in comparison with neutralization activity.

BACKGROUND: Reliable high-throughput serological assays for SARS-CoV-2 antibodies are urgently needed for the effective containment of the COVID-19 pandemic, as it is of crucial importance to understand the strength and duration of immunity after infection, and to make informed decisions concerning the activation or discontinuation of physical distancing restrictions. METHODS: In 184 serum samples from 130 COVID-19 patients and 54 SARS-CoV-2 negative subjects, the analytical and clinical performances of four commercially available chemiluminescent assays (Abbott SARS-Cov-2 IgG, Roche Elecsys anti-SARS-CoV-2, Ortho SARS-CoV-2 total and IgG) and one enzyme-linked immunosorbent assay (Diesse ENZY-WELL SARS-CoV-2 IgG) were evaluated and compared with the neutralization activity achieved using the plaque reduction neutralization test (PRNT). FINDINGS: Precision results ranged from 0.9% to 11.8% for all assays. Elecsys anti-SARS-CoV-2 demonstrated linearity of results at concentrations within the cut-off value. Overall, sensitivity ranged from 78.5 to 87.7%, and specificity, from 97.6 to 100%. On limiting the analysis to samples collected 12 days after onset of symptoms, the sensitivity of all assays increased, the highest value (95.2%) being obtained with VITRO Anti-SARS-CoV-2 Total and Architect SARS-CoV-2 IgG. The strongest PRNT50 correlation with antibody levels was obtained with ENZY-Well SARS-CoV-2 IgG (R2adj = 0.569). INTERPRETATION: The results confirmed that all immunoassays had an excellent specificity, whereas sensitivity varied across immunoassays, depending strongly on the time interval between symptoms onset and sample collection. Further studies should be conducted to achieve a stronger correlation between antibody measurement and PRNT50 in order to obtain useful information for providing a better management of COVID-19 patients, effective passive antibody therapy, and developing a vaccine against the SARS-CoV-2 virus. FUNDING: None.