Coronavirus Antigen Research Articles

Quantification of antibody avidities and accurate detection of SARS-CoV-2 antibodies in serum and saliva on plasmonic substrates.

Accurate assays for the detection of antibodies to SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) are essential for the control of the COVID-19 (coronavirus disease 2019) pandemic. Here, we report antibody and antibody-avidity assays, relying on near-infrared-fluorescence amplification by nanostructured plasmonic gold substrates, for the simultaneous detection of antibodies to the S1 subunit of the spike protein and to the receptor binding domain of SARS-CoV-2 in human serum and saliva, and for quantifying immunoglobulin avidities against coronavirus antigens from SARS-CoV-2, SARS-CoV-1 and the common-cold viruses OC43, HKU1, NL63 and 229E. The antibody assay detected immunoglobulin M in 87% (52 of 60) COVID-19-positive serum samples collected 6 or more days after symptom onset (and the immunoglobulins M and G in all 33 samples collected at least 15 days after symptom onset), and correctly classified 456 out of the 457 COVID-19-negative serum samples tested (424 of them collected before the pandemic, including 73 that were positive for other viruses). We used the antibody-avidity assay to study antibody-maturation patterns, anamnestic responses, and cross-immunity to the common-cold coronaviruses.

First report on Cryptosporidium parvum, Escherichia coli K99, rotavirus and coronavirus in neonatal lambs from north-center region, Algeria

The etiology of neonatal diarrhea is multifactorial and remains one of the greatest health problems in sheep livestock farming. Faecal samples from 559 neonatal lambs aged less than 30 days from 30 sheepfolds located in the north-center region of Algeria were screened with pathogen-specific antigen ELISA for Cryptosporidium parvum, Escherichia coli K99, rotavirus, and coronavirus. Of the 559 lambs, 312 (58.81 %), 155 (27.72 %), 72 (12.88 %) and 20 (3.57 %) were positives for C. parvum, E. coli K99, rotavirus and coronavirus antigens, respectively. The prevalence of C. parvum was the highest (p < 0.0001). C. parvum, E. coli K99, rotavirus and coronavirus were observed in 23 (76.66 %), 17 (56.66 %), 9 (30 %) and 3 (10 %) sheepfolds, respectively. Compared to age, the prevalence of C. parvum was highest during the second and third week of age (p < 0.001). In contrast, other pathogens were found to be more frequent in lambs aged ≤7 days (p < 0.001). The number of lambs with diarrhea was 280 (50.09 %) of which 280 (100 %), 127 (45.35 %), 52 (18.57 %) and 10 (3.57 %) were found to be infected with C. parvum, E. coli K99, rotavirus and coronavirus, respectively (p < 0.0001). In various combinations, mixed infections were detected only with C. parvum. This is the first report of C. parvum, E. coli K99, rotavirus, and coronavirus in ≤30-days old neonatal lambs in Algeria. Special attention should be given to the first colostrum feeding, hygiene of the farm, prevention and control measures for a better prevention of neonatal diarrhea in lambs.

ReScan, a Multiplex Diagnostic Pipeline, Pans Human Sera for SARS-CoV-2 Antigens

SummaryComprehensive understanding of the serological response to SARS-CoV-2 infection is important for both pathophysiologic insight and diagnostic development. Here, we generate a pan-human coronavirus programmable phage display assay to perform proteome-wide profiling of coronavirus antigens enriched by 98 COVID-19 patient sera. Next, we use ReScan, a method to efficiently sequester phage expressing the most immunogenic peptides and print them onto paper-based microarrays using acoustic liquid handling, which isolates and identifies nine candidate antigens, eight of which are derived from the two proteins used for SARS-CoV-2 serologic assays: spike and nucleocapsid proteins. After deployment in a high-throughput assay amenable to clinical lab settings, these antigens show improved specificity over a whole protein panel. This proof-of-concept study demonstrates that ReScan will have broad applicability for other emerging infectious diseases or autoimmune diseases that lack a valid biomarker, enabling a seamless pipeline from antigen discovery to diagnostic using one recombinant protein source.

Detection of feline coronavirus RNA, spike gene mutations, and feline coronavirus antigen in macrophages in aqueous humor of cats in the diagnosis of feline infectious peritonitis.

Uveitis is common in cats, and is often a feature of feline infectious peritonitis (FIP). We evaluated 3 tools for detection of feline coronavirus (FCoV) in aqueous humor: 1) a 7b gene reverse-transcription real-time PCR (7b-RT-rtPCR) assay to detect FCoV RNA, 2) a spike gene mutation RT-rtPCR (S-RT-rtPCR) assay to detect 2 point mutations in the spike gene of FCoV in cats positive by 7b-RT-rtPCR, and 3) immunocytochemistry (ICC) for detection of FCoV antigen in aqueous humor macrophages. We studied 58 cats, including 31 cats with FIP and 27 control cats. FIP was excluded by postmortem examination and negative immunohistochemistry (IHC). Aqueous humor samples obtained postmortem were assessed using 7b-RT-rtPCR in all cats, and positive samples were evaluated with S-RT-rtPCR. ICC evaluation of aqueous humor samples from 36 of the 58 cats was done using an avidin-biotin complex method and monoclonal anti-FCoV IgG 2A. Sensitivity, specificity, and negative and positive predictive values were calculated including 95% CIs. 7b-RT-rtPCR had a specificity of 100.0% (95% CI: 87.2-100.0) and sensitivity of 35.5% (95% CI: 19.2-54.6). Specificity of S-RT-rtPCR could not be determined because there were no FCoV 7b-RT-rtPCR-positive samples in the control group. Sensitivity of S-RT-rtPCR was 12.9% (95% CI 3.6-29.8). Sensitivity and specificity of ICC were 62.5% (95% CI: 40.6-81.2) and 80.0% (95% CI: 44.4-97.5), respectively. The combination of 7b-RT-rtPCR and IHC could be useful in diagnosing FIP; S-RT-rtPCR did not add value; and ICC of aqueous humor samples cannot be recommended for the diagnosis of FIP.

Helen Salisbury: Testing times for GPs.

Patients often turn to their GP for advice on how to stay safe in this uncertain world, to help them apply the rules to their own health condition. They expect us to have privileged access to the latest developments, but in this pandemic we find out about the next policy move only by listening to the news. When it comes to coronavirus antigen testing, GPs have mostly been sitting on the sidelines, frustrated by being unable to request tests for symptomatic patients and not receiving the results of tests done elsewhere. It was announced this week that all patients with symptoms will be able to get an …

Rapid Diagnosis, Isolation, and Treatment of Patients with 2019-Novel Coronavirus Pneumonia: A Retrospective Study of 10-Minute Rapid Test for Coronavirus Antigens

Background: In December, 2019, a novel coronavirus pneumonia (NCP) was first reported in Wuhan, China NCoutbreak spread rapidly nationwide and grobally, Subs

Coronavirus antigen tests: quick and cheap, but too often wrong?

Coronavirus antigen tests: quick and cheap, but too often wrong?

A modular microarray imaging system for highly specific COVID-19 antibody testing.

To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform (TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2–4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100× more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months and years to come to implement efficient containment and mitigation measures, as well as help develop therapeutics and vaccines to treat and prevent the spread of COVID-19.

Clinical evaluation of a rapid diagnostic test kit for detection of canine coronavirus

Canine coronavirus is a single-stranded RNA virus that causes enteritis in dogs of any age. Coronaviral enteritis is seldom definitively diagnosed, since it is usually much less severe than many other types of enteritis and is self-limiting. Conventional diagnostics for the canine coronaviral enteritis such as polymerase chain reaction (PCR), virus isolation, and electron microscopic examination are inappropriate for small animal clinics due to the complicated experimental processes involved. Therefore, a commercially available lateral flow test kit based on chromatographic immunoassay techniques was tested to evaluate its performance as a first-line diagnostic test kit that could be used in clinics. The coronavirus antigen test kit detected canine coronavirus-infected dogs with 93.1% sensitivity and 97.5% specificity. The detection limit of the test kit was between 1.97 × 104/mL and 9.85 × 103/mL for samples with a 2- fold serial dilution from 1.25 × 106 TCID50 (TCID50, 50% tissue culture infectious dose). Additionally, the test kit had no cross-reactivity with canine parvovirus, distemper virus, or Escherichia coli. Overall, the commercially available test kit showed good diagnostic performance in a clinical setting, with results similar to those from PCR, confirming their potential for convenient and accurate use in small animal clinics.

Enteric Pathology of cattle calves naturally infected with corona virus in Jabalpur region

Bovine corona virus (BCV) is one of the most frequently occurring viral agents causing neonatal calf mortality. Rectal faeces collected during necropsy from 38 calves with the history of diarrhoea and gross lesions of enteritis were found positive for Corona virus antigen by ELISA. The tissue samples of intestine, mesenteric lymph nodes and liver collected from eight animals were processed for histopathological studies. Shortening of ileal villi and desquamation of epithelial cells in the lumen were observed in all cases. The colonic mucosa was severely damaged and coated with thick granulocyte rich fibrinopurulent pseudomembrane. Colonic crypts consisted of epithelial cells showing pyknosis, karyorrhexis, granular degeneration, hydropic degeneration, and hyaline droplets. It was concluded that the coronvirus infection mainly affects the ileum and colon in calves leading to diarrhoea and dehydration; its prevalence in calves of Jabalpur region could be an important contributing factor to high incidence of bovine calf mortality in this area.

Bovine gamma/delta T cells are stimulated by bovine coronavirus antigens identified by a soluble T cells receptor

Gamma/delta (γδ) T cells in cattle account for an abundant T cell population. However, little is known regarding the function of γδ T cells as immune cells compared to αβ T cells. Not many pathogen-related antigens have been defined and known to stimulate γδ T cells. To address this information gap, we constructed a soluble receptor for bovine γδ T cells (sγδTCR) that was later used to identify two proteins (156 kDa and 102 kDa) or protein fragments expressed by bovine coronavirus (BCov). The molecular weight of the larger protein suggests it could be the spike glycoprotein of BCov. Subsequently, we used the identified viral proteins to study the reactivity of bovine γδ T cells. In vitro assays showed that purified preparations of the two proteins stimulated WC1+ γδ T cells isolated from cattle. A 4-fold increase in IFN production and a significantly higher expression of MHC class II was observed. However, these viral ligands could not stimulate γδ T cells to synthesize IL-8 or GM-CSF, known to be produced by γδ T cells when stimulated with bacterial antigens. Although the γδ T cells assessed here appeared activated by way of IFN and MHC II expression, surface markers such as CD2, CD25, CD44, CD62L and CD335 were not expressed at significant levels. Further, the activation elicited by viral ligands was not sufficient to induce cytotoxic capability in γδ T cells in vitro as measured by a flow cytometry-based cytotoxicity assay. This in vitro study shows that WC1+ γδ T cells can directly recognize viral antigen

Equine coronavirus: An emerging enteric virus of adult horses.

SummaryEquine coronavirus (ECoV) is an emerging virus associated clinically and epidemiologically with fever, depression, anorexia and less frequently colic and diarrhoea in adult horses. Sporadic cases and outbreaks have been reported with increased frequency since 2010 from Japan, the USA and more recently from Europe. A faeco‐oral transmission route is suspected and clinical or asymptomatic infected horses appear to be responsible for direct and indirect transmission of ECoV. A presumptive clinical diagnosis of ECoV infection may be suggested by clinical presentation, haematological abnormalities such as leucopenia due to lymphopenia and/or neutropenia. Confirmation of ECoV infection is provided by specific ECoV nucleic acid detection in faeces by quantitative PCR (qPCR) or demonstration of coronavirus antigen by immunohistochemistry or electron microscopy in intestinal biopsy material obtained ante or post mortem. The disease is generally self‐limiting and horses typically recover with symptomatic supportive care. Complications associated with disruption of the gastrointestinal barrier have been reported in some infected horses and include endotoxaemia, septicaemia and hyperammonaemia‐associated encephalopathy. Although specific immunoprophylactic measures have been shown to be effective in disease prevention for closely‐related coronaviruses such as bovine coronavirus (BCoV), such strategies have yet not been investigated for horses and disease prevention is limited to basic biosecurity protocols. This article reviews current knowledge concerning the aetiology, epidemiology, clinical signs, diagnosis, pathology, treatment and prevention of ECoV infection in adult horses.

Positive Immunolabelling for Feline Infectious Peritonitis in an African Lion (Panthera leo) with Bilateral Panuveitis

A 15-year-old male African lion (Panthera leo) was presented with blindness due to bilateral panuveitis with retinal detachment. Feline coronavirus (FCoV) antigen was identified immunohistochemically in ocular macrophages, consistent with a diagnosis of feline infectious peritonitis (FIP) infection. This is the first report of FIP in an African lion and the first report of ocular FIP in a non-domestic felid.

Development and validation of a rapid immunochromatographic assay for detection of Middle East respiratory syndrome coronavirus antigen in dromedary camels.

We present here a rapid immunochromatographic assay for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV) antigen in the nasal swabs of dromedary camels. The assay is based on the detection of MERS-CoV nucleocapsid protein in a short time frame using highly selective monoclonal antibodies at room temperature. The relative sensitivity and specificity of the assay were found to be 93.90% and 100%, respectively, compared to that of the UpE and open reading frame 1A (Orf1A) real-time reverse transcriptase PCR (RT-PCR). The results suggest that the assay developed here is a useful tool for the rapid diagnosis and epidemiological surveillance of MERS-CoV infection in dromedary camels.

Severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology.

Although the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) epidemic was controlled by nonvaccine measures, coronaviruses remain a major threat to human health. The design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges: coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathology. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole-virus vaccine candidates alone or adjuvanted with either alum, CpG, or Advax, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addition of adjuvant significantly increased serum neutralizing-antibody titers and reduced lung virus titers on day 3 postchallenge. Whereas unadjuvanted or alum-formulated vaccines were associated with significantly increased lung eosinophilic immunopathology on day 6 postchallenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathology by vaccines containing delta inulin adjuvants correlated better with enhanced T-cell gamma interferon (IFN-γ) recall responses rather than reduced interleukin-4 (IL-4) responses, suggesting that immunopathology predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the critical importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on the ability to induce durable IFN-γ responses. Coronaviruses such as SARS-CoV and Middle East respiratory syndrome-associated coronavirus (MERS-CoV) cause high case fatality rates and remain major human public health threats, creating a need for effective vaccines. While coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology, a problem that is further exacerbated by the formulation of SARS-CoV vaccines with alum adjuvants. This study shows that formulation of SARS-CoV spike protein or inactivated whole-virus vaccines with novel delta inulin-based polysaccharide adjuvants enhances neutralizing-antibody titers and protection against clinical disease but at the same time also protects against development of lung eosinophilic immunopathology. It also shows that immunity achieved with delta inulin adjuvants is long-lived, thereby overcoming the natural tendency for rapidly waning coronavirus immunity. Thus, delta inulin adjuvants may offer a unique ability to develop safer and more effective coronavirus vaccines.

JEPM April 2014—Abstracts

A systemic disease of domestic ferrets characterized by pyogranulomatous inflammation was first recognized in Europe and the United States in 2002. The disease closely resembled feline infectious peritonitis and subsequently has been shown to be associated with ferret systemic coronavirus (FRSCV). A definitive laboratory diagnosis of this disease is typically based on a combination of immunohistochemistry (IHC) and reverse-transcriptase polymerase chain reaction tests to detect FRSCV in granulomatous lesions. In 2010, this feline infectious peritonitis–like disease was first identified in a laboratory ferret in Japan, and laboratory confirmation of the clinical diagnosis was limited to IHC. This report describes 2 cases of systemic coronavirus-associated disease in ferrets presented to Japanese veterinary hospitals. Both presented with pyogranulomatous inflammation in the abdominal cavity, and both cases tested positive for coronavirus antigen by IHC. In 1 case, for which unfixed tissues were available, FRSCV RNA was detected by reverse-transcriptase polymerase chain reaction in the affected tissues.

Two Cases of Systemic Coronavirus-Associated Disease Resembling Feline Infectious Peritonitis in Domestic Ferrets in Japan

A systemic disease of domestic ferrets characterized by pyogranulomatous inflammation was first recognized in Europe and the United States in 2002. The disease closely resembled feline infectious peritonitis and subsequently has been shown to be associated with ferret systemic coronavirus (FRSCV). A definitive laboratory diagnosis of this disease is typically based on a combination of immunohistochemistry (IHC) and reverse-transcriptase polymerase chain reaction tests to detect FRSCV in granulomatous lesions. In 2010, this feline infectious peritonitis–like disease was first identified in a laboratory ferret in Japan, and laboratory confirmation of the clinical diagnosis was limited to IHC. This report describes 2 cases of systemic coronavirus-associated disease in ferrets presented to Japanese veterinary hospitals. Both presented with pyogranulomatous inflammation in the abdominal cavity, and both cases tested positive for coronavirus antigen by IHC. In 1 case, for which unfixed tissues were available, FRSCV RNA was detected by reverse-transcriptase polymerase chain reaction in the affected tissues.

Diagnostic utility of a direct immunofluorescence test to detect feline coronavirus antigen in macrophages in effusive feline infectious peritonitis

The antemortem diagnosis of feline infectious peritonitis (FIP) remains challenging in clinical practice, since current testing methods have suboptimal diagnostic accuracy. Immunohistochemical testing of biopsy specimens and postmortem examination are the standard diagnostic methods, although direct immunofluorescence (DIF) testing to detect feline coronavirus in macrophages in effusion specimens has been reported to have 100% specificity and has been recommended as an antemortem confirmatory test. The aim of this study was to compare the results of DIF testing in antemortem feline effusions with postmortem results using field samples. Effusion specimens were collected antemortem from 17 cats and tested by DIF, followed by postmortem examination. Histopathological examination of specimens collected at postmortem confirmed FIP in 10/17 cases and ruled out FIP out in 7/17 cases. Antemortem DIF testing was positive in all 10 cases confirmed as FIP at postmortem examination. In the seven cats where FIP was ruled out at postmortem examination, DIF was negative in five cases and positive in the remaining two cases. The calculated sensitivity of DIF testing was 100% and the specificity was 71.4%. Duplicate effusion specimens from eight cats that were initially DIF positive were stored refrigerated (4°C) or at room temperature (22–25°C) and subjected to serial DIF testing to determine the duration of positive results. DIF-positive specimens stored at both temperatures retained their positive status for at least 2days.

Positive immunostaining for feline infectious peritonitis (FIP) in a Sphinx cat with cutaneous lesions and bilateral panuveitis

Feline infectious peritonitis (FIP) is a common, fatal, systemic disease of cats. This case report describes the antemortem diagnosis of FIP in a 2‐year‐old spayed female Sphinx cat that presented with a bilateral panuveitis and multiple papular cutaneous lesions. Histopathologically, the skin lesions were characterized by perivascular infiltrates of macrophages, neutrophils, with fewer plasma cells, mast cells, and small lymphocytes in the mid‐ to deep dermis. Immunohistochemistry for intracellular feline coronavirus (FeCoV) antigen demonstrated positive staining in dermal macrophages providing an antemortem diagnosis of a moderate, nodular to diffuse, pyogranulomatous perivascular dermatitis due to FIP infection. Obtaining an antemortem diagnosis of FIP can be a challenge and cutaneous lesions are rare in the disease. Recognition and biopsy of any cutaneous lesions in cats with panuveitis and suspected FIP can help establish an antemortem diagnosis of the disease.

Feline Infectious Peritonitis in a Mountain Lion (Puma concolor), California, USA

Feline infectious peritonitis (FIP) is a fatal immune-mediated vasculitis of felids caused by a mutant form of a common feline enteric virus, feline enteric coronavirus. The virus can attack many organ systems and causes a broad range of signs, commonly including weight loss and fever. Regardless of presentation, FIP is ultimately fatal and often presents a diagnostic challenge. In May 2010, a malnourished young adult male mountain lion (Puma concolor) from Kern County, California, USA was euthanized because of concern for public safety, and a postmortem examination was performed. Gross necropsy and histopathologic examination revealed necrotizing, multifocal myocarditis; necrotizing, neutrophilic, and histiocytic myositis and vasculitis of the tunica muscularis layer of the small and large intestines; and embolic, multifocal, interstitial pneumonia. Feline coronavirus antigen was detected in both the heart and intestinal tissue by immunohistochemistry. A PCR for coronavirus performed on kidney tissue was positive, confirming a diagnosis of FIP. Although coronavirus infection has been documented in mountain lions by serology, this is the first confirmed report of FIP.