Severe Acute Respiratory Syndrome Coronavirus 2 Nucleocapsid Antigen in Urine of Hospitalized Patients With Coronavirus Disease 2019.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid antigen (N-Ag) can be detected in the blood of patients with coronavirus disease 2019 (COVID-19). We used a highly sensitive and specific assay to explore the presence of N-Ag in urine during the course of COVID-19 and its relationship with the severity of disease. METHODS: We studied urinary and plasma N-Ag using a highly sensitive immunoassay in 82 patients with SARS-CoV-2 infection proved by polymerase chain reaction. RESULTS: In the first and second weeks of COVID-19, hospitalized patients tested positive for urinary N-Ag (81.25% and 71.79%, respectively) and plasma N-Ag (93.75% and 94.87%, respectively). High urinary N-Ag levels were associated with the absence of SARS-CoV-2 nucleocapsid antibodies, admission in intensive care units, high C-reactive protein levels, lymphopenia, eosinopenia, and high lactate dehydrogenase levels. Higher accuracy was observed for urinary N-Ag as a predictor of severe COVID-19 than for plasma N-Ag. CONCLUSIONS: Our study demonstrates that N-Ag is present in the urine of patients hospitalized in the early phase of COVID-19. As a direct marker of SARS-CoV-2, urinary N-Ag reflects the dissemination of viral compounds in the body. Urinary N-Ag may be a useful marker for disease severity in SARS-CoV-2 infections.

Lipid and Nucleocapsid N-Protein Accumulation in COVID-19 Patient Lung and Infected Cells.

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global outbreak and prompted an enormous research effort. Still, the subcellular localization of the coronavirus in lungs of COVID-19 patients is not well understood. Here, the localization of the SARS-CoV-2 proteins is studied in postmortem lung material of COVID-19 patients and in SARS-CoV-2-infected Vero cells, processed identically. Correlative light and electron microscopy on semithick cryo-sections demonstrated induction of electron-lucent, lipid-filled compartments after SARS-CoV-2 infection in both lung and cell cultures. In lung tissue, the nonstructural protein 4 and the stable nucleocapsid N-protein were detected on these novel lipid-filled compartments. The induction of such lipid-filled compartments and the localization of the viral proteins in lung of patients with fatal COVID-19 may explain the extensive inflammatory response and provide a new hallmark for SARS-CoV-2 infection at the final, fatal stage of infection. IMPORTANCE Visualization of the subcellular localization of SARS-CoV-2 proteins in lung patient material of COVID-19 patients is important for the understanding of this new virus. We detected viral proteins in the context of the ultrastructure of infected cells and tissues and discovered that some viral proteins accumulate in novel, lipid-filled compartments. These structures are induced in Vero cells but, more importantly, also in lung of patients with COVID-19. We have characterized these lipid-filled compartments and determined that this is a novel, virus-induced structure. Immunogold labeling demonstrated that cellular markers, such as CD63 and lipid droplet marker PLIN-2, are absent. Colocalization of lipid-filled compartments with the stable N-protein and nonstructural protein 4 in lung of the last stages of COVID-19 indicates that these compartments play a key role in the devastating immune response that SARS-CoV-2 infections provoke.

Quantifying SARS-CoV-2 nucleocapsid antigen in oropharyngeal swabs using single molecule array technology.

This study aimed to develop a highly sensitive SARS-CoV-2 nucleocapsid antigen assay using the single molecule array (Simoa) technology and compare it with real time RT-PCR as used in routine clinical practice with the ambition to achieve a comparative technical and clinical sensitivity. Samples were available from 148 SARS-CoV-2 real time RT-PCR positive and 73 SARS-CoV-2 real time RT-PCR negative oropharyngeal swabs. For determination of technical sensitivity SARS-CoV-2 virus culture material was used. The samples were treated with lysis buffer and analyzed using both an in-house and a pre-commercial SARS-CoV-2 nucleocapsid antigen assay on Simoa. Both nucleocapsid antigen assays have a technical sensitivity corresponding to around 100 SARS-CoV-2 RNA molecules/mL. Using a cut-off at 0.1 pg/mL the pre-commercial SARS-CoV-2 nucleocapsid antigen assay had a sensitivity of 96% (95% CI 91.4-98.5%) and specificity of 100% (95% CI 95.1-100%). In comparison the in-house nucleocapsid antigen assay had sensitivity of 95% (95% CI 89.3-98.1%) and a specificity of 100% (95% CI 95.1-100%) using a cut-off at 0.01 pg/mL. The two SARS-CoV-2 nucleocapsid antigen assays correlated with r = 0.91 (P < 0.0001). The in-house and the pre-commercial SARS-CoV-2 nucleocapsid antigen assay demonstrated technical and clinical sensitivity comparable to real-time RT-PCR methods for identifying SARS-CoV-2 infected patients and thus can be used clinically as well as serve as a reference method for antigen Point of Care Testing.

AuNP Coupled Rapid Flow-Through Dot-Blot Immuno-Assay for Enhanced Detection of SARS-CoV-2 Specific Nucleocapsid and Receptor Binding Domain IgG.

BACKGROUND: Serological tests detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are widely used in seroprevalence studies and evaluating the efficacy of the vaccination program. Some of the widely used serological testing techniques are enzyme-linked immune-sorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and lateral flow immunoassay (LFIA). However, these tests are plagued with low sensitivity or specificity, time-consuming, labor-intensive, and expensive. We developed a serological test implementing flow-through dot-blot assay (FT-DBA) for SARS-CoV-2 specific IgG detection, which provides enhanced sensitivity and specificity while being quick to perform and easy to use. METHODS: SARS-CoV-2 antigens were immobilized on nitrocellulose membrane to capture human IgG, which was then detected with anti-human IgG conjugated gold nanoparticle (hIgG-AuNP). A total of 181 samples were analyzed in-house. Within which 35 were further evaluated in US FDA-approved CLIA Elecsys SARS-CoV-2 assay. The positive panel consisted of RT-qPCR positive samples from patients with both <14 days and >14 days from the onset of clinical symptoms. The negative panel contained samples collected from the pre-pandemic era dengue patients and healthy donors during the pandemic. Moreover, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FT-DBA were evaluated against RT-qPCR positive sera. However, the overall efficacies were assessed with sera that seroconverted against either nucleocapsid (NCP) or receptor-binding domain (RBD). RESULTS: In-house ELISA selected a total of 81 true seropositive and 100 seronegative samples. The sensitivity of samples with <14 days using FT-DBA was 94.7%, increasing to 100% for samples >14 days. The overall detection sensitivity and specificity were 98.8% and 98%, respectively, whereas the overall PPV and NPV were 99.6% and 99%. Moreover, comparative analysis between in-house ELISA assays and FT-DBA revealed clinical agreement of Cohen's Kappa value of 0.944. The FT-DBA showed sensitivity and specificity of 100% when compared with commercial CLIA kits. CONCLUSION: The assay can confirm past SARS-CoV-2 infection with high accuracy within 2 minutes compared to commercial CLIA or in-house ELISA. It can help track SARS-CoV-2 disease progression, population screening, and vaccination response. The ease of use of the assay without requiring any instruments while being semi-quantitative provides the avenue of its implementation in remote areas around the globe, where conventional serodiagnosis is not feasible.

Performance evaluation of an automated SARS-CoV-2 Ag test for the diagnosis of COVID-19 infection on nasopharyngeal swabs.

OBJECTIVES: The detection of SARS-CoV-2 in infected people is a key tool to help in controlling COVID-19 pandemic. Like rapid antigenic tests, automated antigen tests, that present the advantage of a higher throughput flow, may be of interest. The LIAISON® SARS-CoV-2 Ag test was evaluated for the quantification of SARS-CoV-2 nucleocapsid antigen in nasopharyngeal swabs by comparison to RT-PCR. METHODS: The study involved 378 nasopharyngeal samples (UTM® and FLOQSwab™, Copan Diagnostics), including 46 swabs positive for SARS-CoV-2 by RT-PCR. These samples came from asymptomatic (n=99, 26.2%) or symptomatic people (n=279, 73.8%), at different times from symptom onset. The samples were analyzed on LIAISON® XL. RESULTS: The overall specificity was 99.4% (CI95% [98.6-100]). The negative predictive value reached 100% in asymptomatic people. Among the 46 positive samples, the overall sensitivity was 84.8% (CI95% [74.4-95.2]), reached 91.9% (CI95% [83.1-100]) in the first fourth days after symptoms onset and was 100% for Cq values ≤25. Antigen was not detected in samples with Cq values >25. Similar results were observed on nasopharyngeal swabs coming from patients infected with the 20I/501Y.V1 variant or the 20H/501Y.V2 variant. CONCLUSIONS: According to technical performances, the LIAISON® SARS-CoV-2 Ag test may be a useful tool for COVID-19 diagnosis, especially during the first four days of symptoms.

Improved Detection Sensitivity of an Antigen Test for SARS-CoV-2 Nucleocapsid Proteins with Thio-NAD Cycling.

Antigen tests for infectious diseases are inexpensive and easy-to-use, but the limit of detection (LOD) is generally higher than that of PCR tests, which are considered the gold standard. In the present study, we combined a sandwich enzyme-linked immunosorbent assay (ELISA) with thionicotinamide-adenine dinucleotide (thio-NAD) cycling to improve the LOD of antigen tests for coronavirus disease 2019 (COVID-19). For recombinant nucleocapsid proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the LOD of our ELISA with thio-NAD cycling was 2.95 × 10-17 moles/assay. When UV-irradiated inactive SARS-CoV-2 was used, the minimum detectable virions corresponding to 2.6 × 104 RNA copies/assay were obtained using our ELISA with thio-NAD cycling. The assay volume for each test was 100 µL. The minimum detectable value was smaller than that of the latest antigen test using a fluorescent immunoassay for SARS-CoV-2, indicating the validity of our detection system for COVID-19 diagnosis.

Molecular determinants of Ebola nucleocapsid stability from molecular dynamics simulations.

Ebola virus (EBOV) is a human pathogen with the ability to cause hemorrhagic fever and bleeding diathesis in hosts. The life cycle of EBOV depends on its nucleocapsid. The Ebola nucleocapsid consists of a helical assembly of nucleoproteins (NPs) encapsidating single-stranded viral RNA (ssRNA). Knowledge of the molecular determinants of Ebola nucleocapsid stability is essential for the development of therapeutics against EBOV. However, large degrees of freedom associated with the Ebola nucleocapsid helical assembly pose a computational challenge, thereby limiting the previous simulation studies to the level of monomers. In the present work, we have performed all atom molecular dynamics (MD) simulations of the helical assembly of EBOV nucleoproteins in the absence and presence of ssRNA. We found that ssRNA is essential for maintaining structural integrity of the nucleocapsid. Other molecular determinants observed to stabilize the nucleocapsid include NP-RNA and NP-NP interactions and ion distributions. Additionally, the structural and dynamical behavior of the nucleocapsid monomer depends on its position in the helical assembly. NP monomers present on the longitudinal edges of the helical tube are more exposed, flexible, and have weaker NP-NP interactions than those residing in the center. This work provides key structural features stabilizing the nucleocapsid that may serve as therapeutic targets.

A simple, high-throughput stabilization assay to test HIV-1 uncoating inhibitors.

Shortly after entering the cell, HIV-1 copies its genomic RNA into double-stranded DNA in a process known as reverse transcription. This process starts inside a core consisting of an enclosed lattice of capsid proteins that protect the viral RNA from cytosolic sensors and degradation pathways. To accomplish reverse transcription and integrate cDNA into the host cell genome, the capsid shell needs to be disassembled, or uncoated. Premature or delayed uncoating attenuates reverse transcription and blocks HIV-1 infectivity. Small molecules that bind to the capsid lattice of the HIV-1 core and either destabilize or stabilize its structure could thus function as effective HIV-1 inhibitors. To screen for such compounds, we modified our recently developed FAITH assay to allow direct assessment of the stability of in vitro preassembled HIV-1 capsid-nucleocapsid (CANC) tubular particles. This new assay is a high-throughput fluorescence method based on measuring the amount of nucleic acid released from CANC complexes under disassembly conditions. The amount of disassembled CANC particles and released nucleic acid is proportional to the fluorescence signal, from which the relative percentage of CANC stability can be calculated. We consider our assay a potentially powerful tool for in vitro screening for compounds that alter HIV disassembly.

Generation, identification, and functional analysis of monoclonal antibodies against porcine epidemic diarrhea virus nucleocapsid.

The variant strains of porcine epidemic diarrhea virus (PEDV) severely threaten the pig industry worldwide and cause up to 100% mortality in suckling piglets. It is critically important and urgent to develop tools for detection of PEDV infection. In this study, we developed six monoclonal antibodies (mAbs) targeting N protein of PEDV and analyzed their applications on enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), western blot assay, and flow cytometry assay. The results demonstrated that all these six mAbs were IgG1 isotype and κ chain. Among these six mAbs, 3F12 recognizes a linear epitope (VAAVKDALKSLGI) while the other five mAbs recognize different conformational epitopes formed by a specific peptide fragment or the full length of N protein. The functional analysis showed that all these six mAbs were applicable to ELISA, western blot, IFA, and flow cytometry assay. In conclusion, we developed six mAbs against PEDV-N protein to facilitate the early detection of PEDV infection using ELISA, western blot, IFA, and flow cytometry.

Ultrastructural analysis of virion formation and anterograde intraaxonal transport of the alphaherpesvirus pseudorabies virus in primary neurons.

A hallmark of alphaherpesviruses is their capacity to be neuroinvasive and establish latent infections in neurons. After primary replication in epithelial cells at the periphery, entry into nerve endings occurs, followed by retrograde transport of nucleocapsids to the nucleus where viral transcription, genome replication, and nucleocapsid formation take place. Translocation of nucleocapsids to the cytoplasm is followed by axonal transport to infect synaptically linked neurons. Two modes of intraaxonal anterograde herpesvirus transport have been proposed: transport of complete, enveloped virions within vesicles ("married model"), and separate transport of capsids and envelopes ("subassembly model"). To assess this in detail for the alphaherpesvirus pseudorabies virus (PrV), we used high-resolution transmission electron microscopy of primary neuronal cultures from embryonic rat superior cervical ganglia after infection with wild-type and gB-deficient PrV. Our data show that intranuclear capsid maturation, nuclear egress and cytoplasmic secondary envelopment occur as in cultured nonpolarized cells (H. Granzow, F. Weiland, A. Jöns, B. G. Klupp, A. Karger, and T. C. Mettenleiter, J. Virol. 71:2072-2082, 1997). PrV virions were present in axons as enveloped particles within vesicles associated with microtubules and apparently leave the neuron by exocytosis primarily at the growth cone. Only a few nonenveloped nucleocapsids were found in the axon. The same picture was observed after infection by phenotypically complemented gB-deficient PrV, which is able to complete only a single round of replication. Our data thus support intraaxonal anterograde transport of enveloped PrV virions within vesicles following the "married model."

Rapid detection of bovine coronavirus by a semi-nested RT-PCR / Detecção rápida do Coronavírus Bovino (BCoV) por meio de uma semi-nested RT-PCR

Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.

O Coronavírus bovino (BCoV) pertence ao grupo 2 do gênero Coronavirus (Nidovirales: Coronaviridae) e é agente causador de enterites tanto em bezerros como em bovinos adultos, bem como de doença respiratória em bezerros. O presente estudo teve por objetivo desenvolver uma semi-nested RT-PCR para a detecção do BCoV com base em seqüências representativas e recentes do gene do nucleocapsídeo, região conservada do genoma dos coronavírus. Três primers foram desenhados, a primeira amplificação com um fragmento esperado de 463pb e a segunda (semi-nested) com um fragmento esperado de 306pb. A sensibilidade analítica foi determinada pela diluição do BCoV cepa Kakegawa (título HA: 256) na base de 10 em água ultra-pura tratada com DEPC, em soro fetal bovino (SFB) e em uma suspensão fecal negativa para o BCoV, onde foram encontrados resultados positivos até a diluição de 10-2, 10-3 e 10-7, respectivamente. Este resultado sugere que a quantidade total de RNA na amostra influencia na precipitação dos pellets pelo método de extração utilizado. Quando se utiliza amostra fecal, a grande quantidade de RNA total funciona como carreadora do RNA do BCoV, demonstrando elevada sensibilidade analítica e ausência de possíveis substâncias inibidoras da PCR. O protocolo final da semi-nested RT-PCR foi aplicado a 25 amostras fecais de vacas adultas, previamente avaliadas por uma nested RT-PCR RdRp utilizada como teste de referência, resultando em 20 e 17 amostras positivas para o primeiro e segundo teste, respectivamente. Os resultados dos dois sistema de diagnóstico apresentaram concordância substancial (kappa: 0,694). A elevada sensibilidade e especificidade do novo método proposto e o fato de que os primers foram desenhados baseados em sequências atuais do BCoV, oferecem bases para o diagnóstico mais acurado de infecções causadas pelo BCoV, assim como para novas perspectivas em protocolos de detecção de outros Coronavírus de importância tanto em saninade animal ...

WELYSSA: a simple tool using mouse monoclonal antibodies for the detection of lyssavirus nucleocapsid in rabies suspected specimens.

A Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assay (ELISA) named WELYSSA was developed for the diagnosis of rabies suspected specimens using prototype viruses from the different genotypes of lyssavirus and from various geographic origins and phylogenetic lineages. It included a panel of 1,660 specimens received for rabies diagnostic testing, and was found to be highly specific (99.9%) and sensitive (97.0%) when compared to other recommended rabies diagnostic methods.

A simple sandwich ELISA (WELYSSA) for the detection of lyssavirus nucleocapsid in rabies suspected specimens using mouse monoclonal antibodies.

Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISA) were developed for the diagnosis of rabies-suspect specimens. A combination of four mouse monoclonal antibodies directed against the rabies virus nucleocapsid was selected and used for the detection. The test was optimized and standardized so that maximum concordance could be maintained with the standard procedures of rabies diagnosis recommended by the WHO expert committee. Using prototype viruses from the different genotypes of lyssavirus and from various geographic origins and phylogenetic lineages, this paper presents a reliable, rapid and transferable diagnostic method, named WELYSSA that readily permits the detection of lyssaviruses belonging to the 7 genotypes of lyssavirus circulating in Europe, Africa, Asia and Oceania. The threshold of detection of lyssavirus nucleocapsids is low (0.8 ng/ml). With a panel of 1030 specimens received for rabies diagnostic testing, this test was found to be highly specific (0.999) and sensitive (0.970) when compared to other recommended rabies diagnostic methods.

Novel rapid immunochromatographic test based on an enzyme immunoassay for detecting nucleocapsid antigen in SARS-associated coronavirus.

A novel severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been discovered. The detection of both antigens and antibodies in SARS-CoV from human specimens with suspected SARS plays an important role in preventing infection. We developed a novel rapid immunochromatographic test (RICT) based on the sandwich format enzyme immunoassay (EIA) with an all-in-one device for detecting the native nucleocapsid antigen (N-Ag) of SARS-CoV using monoclonal antibodies (MoAbs), which we produced by immunizing recombinant N-Ag to mice. RICT is a qualitative assay for respiratory aspirates and serum specimens. With this assay, a positive result can be judged subjectively by the appearance of a blue line on the device 15 min after the sample is applied. RICT with several pairs of MoAbs showed a high sensitivity for the detection of recombinant N-Ag as well as viral N-Ag of SARS-CoV. rSN122 and rSN21-2 were the best MoAbs for immobilized antibody and enzyme labeling, respectively. With regard to analytical sensitivity, RICT detected N-Ag at 31 pg/mL for recombinant N-Ag, and at 1.99 x 10(2) TCID(50)/mL for SARS-CoV. The specificity of RICT was 100% when 150 human sera and 50 nasopharyngeal aspirates (NSPs) were used. RICT based on an EIA using the rSN122/rSN21-2 pair is a sensitive, specific, and reliable rapid assay for detecting N-Ag in SARS-CoV treated with either heat or Triton X-100.

Immunofluorescence assay for detection of the nucleocapsid antigen of the severe acute respiratory syndrome (SARS)-associated coronavirus in cells derived from throat wash samples of patients with SARS.

An antigen detection assay for severe acute respiratory syndrome (SARS) coronavirus was established in this study by an indirect immunofluorescence test, which utilized cells derived from throat wash samples of patients with SARS and a rabbit serum that recognized the nucleocapsid protein of SARS-associated coronavirus (SARS-CoV) but not that of other human coronavirus tested. It detected SARS-CoV in 11 of 17 (65%) samples from SARS patients as early as day 2 of illness but in none of the 10 samples from healthy controls. Compared with other diagnostic modalities for detecting SARS-CoV, this assay is simpler, more convenient, and economical. It could be an alternative for early and rapid diagnosis, should SARS return in the future.

Use of the COOH portion of the nucleocapsid protein in an antigen-capturing enzyme-linked immunosorbent assay for specific and sensitive detection of severe acute respiratory syndrome coronavirus.

Antibody detection with a recombinant COOH portion of the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid (N) protein, N13 (amino acids 221 to 422), was demonstrated to be more specific and sensitive than that with the full-length N protein, and an N13-based antigen-capturing enzyme-linked immunosorbent assay providing a convenient and specific test for serodiagnosis and epidemiological study of SARS was developed.

Evaluation of a peptide-based enzyme immunoassay for anti-SARS coronavirus IgG antibody.

High throughput assays for anti-SARS-CoV IgG antibody detection are need for large-scale epidemiologic studies. The performance of a microplate enzyme immunoassay, DETECT-SARS was evaluated for the detection of anti-SARS-CoV IgG antibody. This assay is based on synthetic peptides derived from the nucleocapsid and spike proteins. The results showed that the assay provided a high degree of sensitivity (95.9%) for convalescent serum samples. The level of specificity was close to 90%, and did not show significant variation among different control groups. The high degree of sensitivity together with the high-throughput nature makes it advantageous as a screening assay for studies where handling of a large number of specimens is required.

[Detection of RNA of SARS coronavirus in hospital sewage].

OBJECTIVE: In order to explore the existence of SARS coronavirus (Co-V) and/or its RNA in sewage of hospitals administered SARS patients. METHODS: A novel electropositive filter was used to concentrate the SARS-CoV from the sewage of two hospitals administered SARS patients in Beijing, including twelve 2,500 ml sewage samples from the hospitals before disinfection, and ten 25,000 ml samples after disinfection; as well as cell culture, RT-PCR and sequencing of gene to detect and identify the viruses from sewage. RESULTS: There was no live SARS-CoV detected in the sewage in this study. The nucleic acid of SARS-CoV had been found in the 12 sewage samples before disinfection from both hospitals by semi-nested PCR. After disinfection, SARS-CoV RNA could only be detected from the samples from the 309th Hospital, and the others were negative. CONCLUSION: It provides evidence that there is no live SARS-Cov in the sewage from hospitals with SARS patients though SARS-CoV RNA can be detected.

Detection of severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein in sars patients by enzyme-linked immunosorbent assay.

We report the development of an enzyme-linked immunosorbent assay (ELISA) for the detection of severe acute respiratory syndrome (SARS) coronavirus (CoV) nucleocapsid protein. The assay was carried out with hyperimmune polyclonal nucleocapsid-specific antibodies from guinea pigs and rabbits immunized with recombinant His(6)-tagged SARS CoV nucleocapsid protein. The assay was used for the detection of SARS CoV nucleocapsid protein in nasopharyngeal aspirate, urine, and fecal samples collected from patients with confirmed SARS between days 2 and 33 after the onset of illness. The ELISA was capable of detecting this protein in SARS CoV cell culture lysates at 15 50% tissue culture infective doses/ml but did not produce positive signals when tested with cell culture lysates of human coronaviruses OC43 and 229E. When tested with 120 nasopharyngeal aspirate, 100 urine, and 100 fecal specimens from hospitalized patients without SARS, the assay was shown to have high specificities-96.7, 99, and 96%, respectively. In an evaluation of clinical specimens from SARS patients, 34 (52%) of 66 nasopharyngeal aspirate samples from 50 patients, 5 (5%) of 94 urine samples from 94 patients, and 36 (55%) of 65 fecal samples from 65 patients tested positive for SARS CoV nucleocapsid protein. Nucleocapsid protein could be detected from days 6 to 24 in nasopharyngeal aspirate specimens, from days 11 to 31 in urine specimens, and from days 8 to 32 in fecal specimens after the onset of illness. Moreover, the protein could be detected in 25 (83%) of 30 nasopharyngeal aspirate specimens obtained from days 11 to 15 and in all 7 fecal specimens obtained from days 21 to 32. Since the present ELISA is more convenient and economical than reverse transcription-PCR, it may serve as an alternative tool for the early diagnosis of SARS CoV infection in laboratories with limited resources and expertise and for mass screening for the reservoir of SARS CoV. Further studies on serial clinical specimens should reveal the duration of nucleocapsid protein shedding and may reveal a higher detection rate in SARS patients.

[Expression of the monoclonal antibody against nucleocapsid antigen of SARS-associated coronavirus in autopsy tissues from SARS patients].

OBJECTIVE: To investigate the presence and distribution of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) in autopsy tissues obtained from patients died of SARS. METHODS: Immunohistochemical technique was applied in 4 fatal SARS cases to examine the autopsy tissues including the lungs, spleen, lymph nodes, brain, pituitary, heart, liver, kidney, pancreas, trachea, esophagus, gastrointestinal tract, adrenal glands, parathyroids, skin and bone marrow. RESULTS: Immunohistochemistry identified positive monoclonal antibody against SARS-CoV nuceeocapsid (N) protein in the alveolar epithelium and the infiltrating monocytes or macrophages in the lung, spleen and lymph nodes; the presence of the antibody was also detected in the serous gland epithelium of the trachea/bronchus, squamous epithelium of the esophagus, the gastric parietal cells, the epithelium of the intestinal tract, acidophilic cells in the parathyroids and pituitary, acinus cells in the pancreas, adrenal cortical cells, sweat gland cells, small vessel endothelium, bone marrow promyelocytes, epithelial cells of the distal convoluted tubule of the kidney, brain neurons, and the hepatocytes near the central vein. CONCLUSIONS: A variety of organs and tissues can be infected by SARS-CoV, and the positive expression of SARS-CoV N protein in the epithelial cells of the gastrointestinal tract, the distal convoluted tubule of the kidney and the sweat gland cells is significant for studying the transmission routes of SARS.