Longitudinal analysis of SARS-CoV-2 infection and vaccination in the LA-SPARTA cohort reveals increased risk of infection in vaccinated Hispanic participants.

Introduction: SARS-CoV-2 is the etiologic agent of coronavirus disease 2019 (COVID-19). Questions remain regarding correlates of risk and immune protection against COVID-19. Methods: We prospectively enrolled 200 participants with a high risk of SARS-CoV-2 occupational exposure at a U.S. medical center between December 2020 and April 2022. Participant exposure risks, vaccination/infection status, and symptoms were followed longitudinally at 3, 6, and 12 months, with blood and saliva collection. Serological response to the SARS-CoV-2 spike holoprotein (S), receptor binding domain (RBD) and nucleocapsid proteins (NP) were quantified by ELISA assay. Results: Based on serology, 40 of 200 (20%) participants were infected. Healthcare and non-healthcare occupations had equivalent infection incidence. Only 79.5% of infected participants seroconverted for NP following infection, and 11.5% were unaware they had been infected. The antibody response to S was greater than to RBD. Hispanic ethnicity was associated with 2-fold greater incidence of infection despite vaccination in this cohort. Discussion: Overall, our findings demonstrate: 1) variability in the antibody response to SARS-CoV-2 infection despite similar exposure risk; 2) the concentration of binding antibody to the SARS-CoV-2 S or RBD proteins is not directly correlated with protection against infection in vaccinated individuals; and 3) determinants of infection risk include Hispanic ethnicity despite vaccination and similar occupational exposure.

Breakthrough infections with the omicron and delta variants of SARS-CoV-2 result in similar re-activation of vaccine-induced immunity.

Background: Results showing that sera from double vaccinated individuals have minimal neutralizing activity against Omicron have been interpreted as indicating the need for a third vaccine dose for protection. However, there is little information about early immune responses to Omicron infection in double vaccinated individuals. Methods: We measured inflammatory mediators, antibodies to the SARS-CoV-2 spike and nucleocapsid proteins, and spike peptide-induced release of interferon gamma in whole blood in 51 double-vaccinated individuals infected with Omicron, in 14 infected with Delta, and in 18 healthy controls. The median time points for the first and second samples were 7 and 14 days after symptom onset, respectively. Findings: Infection with Omicron or Delta led to a rapid and similar increase in antibodies to the receptor-binding domain (RBD) of Omicron protein and spike peptide-induced interferon gamma in whole blood. Both the Omicron- and the Delta-infected patients had a mild and transient increase in inflammatory parameters. Interpretation: The results suggest that two vaccine doses are sufficient to mount a rapid and potent immune response upon infection in healthy individuals of with the Omicron variant. Funding: The study was funded by the Oslo University Hospital, and by grants from The Coalition for Epidemic Preparedness Innovations, Research Council of Norway (no 312780, 324272), South-Eastern Norway Regional Health Authority (no 2019067, 2021071, 10357, 2021047, 33612, 2021087, 2017092), EU Horizon 2020 grant no 848099, a philantropic donation from Vivaldi Invest A/S, and The European Virus Archive Global.

Association between IgG responses against the nucleocapsid proteins of alphacoronaviruses and COVID-19 severity.

Understanding immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to contain the COVID-19 pandemic. Using a multiplex approach, serum IgG responses against the whole SARS-CoV-2 proteome and the nucleocapsid proteins of endemic human coronaviruses (HCoVs) were measured in SARS-CoV-2-infected donors and healthy controls. COVID-19 severity strongly correlated with IgG responses against the nucleocapsid (N) of SARS-CoV-2 and possibly with the number of viral antigens targeted. Furthermore, a strong correlation between COVID-19 severity and serum responses against N of endemic alpha- but not betacoronaviruses was detected. This correlation was neither caused by cross-reactivity of antibodies, nor by a general boosting effect of SARS-CoV-2 infection on pre-existing humoral immunity. These findings raise the prospect of a potential disease progression marker for COVID-19 severity that allows for early stratification of infected individuals.

Low complexity domains of the nucleocapsid protein of SARS-CoV-2 form amyloid fibrils.

The self-assembly of the Nucleocapsid protein (NCAP) of SARS-CoV-2 is crucial for its function. Computational analysis of the amino acid sequence of NCAP reveals low-complexity domains (LCDs) akin to LCDs in other proteins known to self-assemble as phase separation droplets and amyloid fibrils. Previous reports have described NCAP's propensity to phase-separate. Here we show that the central LCD of NCAP is capable of both, phase separation and amyloid formation. Within this central LCD we identified three adhesive segments and determined the atomic structure of the fibrils formed by each. Those structures guided the design of G12, a peptide that interferes with the self-assembly of NCAP and demonstrates antiviral activity in SARS-CoV-2 infected cells. Our work, therefore, demonstrates the amyloid form of the central LCD of NCAP and suggests that amyloidogenic segments of NCAP could be targeted for drug development.

Antibody Response to the SARS-CoV-2 Spike and Nucleocapsid Proteins in Patients with Different COVID-19 Clinical Profiles.

The first case of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in Brazil was diagnosed on February 26, 2020. Due to the important epidemiological impact of COVID-19, the present study aimed to analyze the specificity of IgG antibody responses to the S1, S2 and N proteins of SARS-CoV-2 in different COVID-19 clinical profiles. This study enrolled 136 individuals who were diagnosed with or without COVID-19 based on clinical findings and laboratory results and classified as asymptomatic or as having mild, moderate or severe disease. Data collection was performed through a semistructured questionnaire to obtain demographic information and main clinical manifestations. IgG antibody responses to the S1 and S2 subunits of the spike (S) protein and the nucleocapsid (N) protein were evaluated using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. The results showed that among the participants, 87.5% (119/136) exhibited IgG responses to the S1 subunit and 88.25% (120/136) to N. Conversely, only 14.44% of the subjects (21/136) displayed S2 subunit responses. When analyzing the IgG antibody response while considering the different proteins of the virus, patients with severe disease had significantly higher antibody responses to N and S1 than asymptomatic individuals (p ≤ 0.0001), whereas most of the participants had low antibody titers against the S2 subunit. In addition, individuals with long COVID-19 showed a greater IgG response profile than those with symptomatology of a short duration. Based on the results of this study, it is concluded that levels of IgG antibodies may be related to the clinical evolution of COVID-19, with high levels of IgG antibodies against S1 and N in severe cases and in individuals with long COVID-19.

SARS-CoV-2 antibody responses associate with sex, age and disease severity in previously uninfected people admitted to hospital with COVID-19: An ISARIC4C prospective study.

The SARS-CoV-2 pandemic enables the analysis of immune responses induced against a novel coronavirus infecting immunologically naïve individuals. This provides an opportunity for analysis of immune responses and associations with age, sex and disease severity. Here we measured an array of solid-phase binding antibody and viral neutralising Ab (nAb) responses in participants (n=337) of the ISARIC4C cohort and characterised their correlation with peak disease severity during acute infection and early convalescence. Overall, the responses in a Double Antigen Binding Assay (DABA) for antibody to the receptor binding domain (anti-RBD) correlated well with IgM as well as IgG responses against viral spike, S1 and nucleocapsid protein (NP) antigens. DABA reactivity also correlated with nAb. As we and others reported previously, there is greater risk of severe disease and death in older men, whilst the sex ratio was found to be equal within each severity grouping in younger people. In older males with severe disease (mean age 68 years), peak antibody levels were found to be delayed by one to two weeks compared with women, and nAb responses were delayed further. Additionally, we demonstrated that solid-phase binding antibody responses reached higher levels in males as measured via DABA and IgM binding against Spike, NP and S1 antigens. In contrast, this was not observed for nAb responses. When measuring SARS-CoV-2 RNA transcripts (as a surrogate for viral shedding) in nasal swabs at recruitment, we saw no significant differences by sex or disease severity status. However, we have shown higher antibody levels associated with low nasal viral RNA indicating a role of antibody responses in controlling viral replication and shedding in the upper airway. In this study, we have shown discernible differences in the humoral immune responses between males and females and these differences associate with age as well as with resultant disease severity.

Validation of N Protein Antibodies to Diagnose Previous SARS-CoV-2 Infection in a Large Cohort of Healthcare Workers: Use of Roche Elecsys® Immunoassay in the S Protein Vaccination Era.

The aim of this study was to validate the detection of anti-nucleocapsid protein (N protein) antibodies for the diagnosis of SARS-CoV-2 infection in light of the fact that most COVID-19 vaccines use the spike (S) protein as the antigen. Here, 3550 healthcare workers (HCWs) were enrolled from May 2020 (when no S protein vaccines were available). We defined SARS-CoV-2 infection if HCWs were found to be positive by RT-PCR or found to be positive in at least two different serological immunoassays. Serum samples from Biobanc I3PT-CERCA were analyzed by Roche Elecsys® (N protein) and Vircell IgG (N and S proteins) immunoassays. Discordant samples were reanalyzed with other commercial immunoassays. Roche Elecsys® showed the positivity of 539 (15.2%) HCWs, 664 (18.7%) were found to be positive by Vircell IgG immunoassays, and 164 samples (4.6%) showed discrepant results. According to our SARS-CoV-2 infection criteria, 563 HCWs had SARS-CoV-2 infection. The Roche Elecsys® immunoassay has a sensitivity, specificity, accuracy, and concordance with the presence of infection of 94.7%, 99.8%, 99.3%, and 0.96, respectively. Similar results were observed in a validation cohort of vaccinated HCWs. We conclude that the Roche Elecsys® SARS-CoV-2 N protein immunoassay demonstrated good performance in diagnosing previous SARS-CoV-2 infection in a large cohort of HCWs.

Phase-separated nucleocapsid protein of SARS-CoV-2 suppresses cGAS-DNA recognition by disrupting cGAS-G3BP1 complex.

Currently, the incidence and fatality rate of SARS-CoV-2 remain continually high worldwide. COVID-19 patients infected with SARS-CoV-2 exhibited decreased type I interferon (IFN-I) signal, along with limited activation of antiviral immune responses as well as enhanced viral infectivity. Dramatic progresses have been made in revealing the multiple strategies employed by SARS-CoV-2 in impairing canonical RNA sensing pathways. However, it remains to be determined about the SARS-CoV-2 antagonism of cGAS-mediated activation of IFN responses during infection. In the current study, we figure out that SARS-CoV-2 infection leads to the accumulation of released mitochondria DNA (mtDNA), which in turn triggers cGAS to activate IFN-I signaling. As countermeasures, SARS-CoV-2 nucleocapsid (N) protein restricts the DNA recognition capacity of cGAS to impair cGAS-induced IFN-I signaling. Mechanically, N protein disrupts the assembly of cGAS with its co-factor G3BP1 by undergoing DNA-induced liquid-liquid phase separation (LLPS), subsequently impairs the double-strand DNA (dsDNA) detection ability of cGAS. Taken together, our findings unravel a novel antagonistic strategy by which SARS-CoV-2 reduces DNA-triggered IFN-I pathway through interfering with cGAS-DNA phase separation.

A Multiplexed SERS Microassay for Accurate Detection of SARS-CoV-2 and Variants of Concern.

Severe acute respiratory syndrome coronavirus 2 variants play an important role in predicting patient outcome during postinfection, and with growing fears of COVID-19 reservoirs in domestic and wild animals, it is necessary to adapt detection systems for variant detection. However, variant-specific detection remains challenging. Surface-enhanced Raman scattering is a sensitive and multiplexing technique that allows the simultaneous detection of multiple targets for accurate identification. Here we propose the development of a multiplex SERS microassay to detect both the spike and nucleocapsid structural proteins of SARS-CoV-2. The designed SERS microassay integrates gold-silver hollow nanobox barcodes and electrohydrodynamically induced nanomixing which in combination enables highly specific and sensitive detection of SARS-CoV-2 and the S-protein epitopes to delineate between ancestral prevariant strains with the newer variants of concern, Delta and Omicron. The microassay allows detection from as low as 20 virus/µL and 50 pg/mL RBD protein and can clearly identify the virus among infected versus healthy nasopharyngeal swabs, with the potential to identify between variants. The detection of both S- and N-proteins of SARS-CoV-2 and the differentiation of variants on the SERS microassay can aid the early detection of COVID-19 to reduce transmission rates and lead into adequate treatments for those severely affected by the virus.

Suramin Disturbs the Association of the N-Terminal Domain of SARS-CoV-2 Nucleocapsid Protein with RNA.

Suramin was originally used as an antiparasitic drug in clinics. Here, we demonstrate that suramin can bind to the N-terminal domain of SARS-CoV-2 nucleocapsid protein (N-NTD) and disturb its interaction with RNA. The BLI experiments showed that N-NTD interacts suramin with a dissociate constant (Kd = 2.74 µM) stronger than that of N-NTD with ssRNA-16 (Kd = 8.37 µM). Furthermore, both NMR titration experiments and molecular docking analysis suggested that suramin mainly binds to the positively charged cavity between the finger and the palm subdomains of N-NTD, and residues R88, R92, R93, I94, R95, K102 and A156 are crucial for N-NTD capturing suramin. Besides, NMR dynamics experiments showed that suramin-bound N-NTD adopts a more rigid structure, and the loop between ß2-ß3 exhibits fast motion on the ps-ns timescale, potentially facilitating suramin binding. Our findings not only reveal the molecular basis of suramin disturbing the association of SARS-CoV-2 N-NTD with RNA but also provide valuable structural information for the development of drugs against SARS-CoV-2.

TAT Nanobody Exerts Antiviral Effect against PRRSV In Vitro by Targeting Viral Nucleocapsid Protein.

Porcine reproductive and respiratory syndrome (PRRS) is caused by the PRRS virus (PRRSV), which has brought huge economic losses to the pork industry worldwide since its first discovery in the late 1980s in North America. To date, there are no effective commercial vaccines or therapeutic drugs available for controlling the spread of PRRSV. Due to their unique advantages of high affinity and high specificity, nanobodies (Nbs) have received increasing attention in the process of disease diagnosis and treatment. Trans-activator transcription (TAT) can serve as a vector to carry specific proteins into cells by passing through cell membranes. In our previous study, a specific Nb against the PRRSV nucleocapsid (N) protein was screened using phage display technology. For this study, we developed a novel recombinant protein constituting a TAT-conjugated Nb, which we call TAT-Nb1. The target cell entry efficiency of TAT-Nb1 and its effect on PRRSV infection and replication were then investigated. Our results indicate that TAT delivered Nb1 into Marc-145 cells and porcine alveolar macrophages (PAMs) in a dose- and time-dependent manner. Furthermore, TAT-Nb1 dose-dependently suppressed PRRSV infection and replication, where this antiviral effect was independent of PRRSV strain. Co-immunoprecipitation results revealed that Nb1 efficiently interacted with the N protein of PRRSV. Taken together, the presented results suggest that TAT-Nb1 can effectively suppress PRRSV replication, and it may be considered as a new anti-PRRSV candidate drug.

Screen for Modulation of Nucleocapsid Protein Condensation Identifies Small Molecules with Anti-Coronavirus Activity.

Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase-separated condensates that are hypothesized to play critical roles in viral replication, transcription, and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species. Here, we show that N proteins from all seven human coronaviruses (HCoVs) vary in their tendency to undergo phase separation when expressed in human lung epithelial cells. We developed a cell-based high-content screening platform and identified small molecules that both promote and inhibit condensation of SARS-CoV-2 N. Interestingly, these host-targeted small molecules exhibited condensate-modulatory effects across all HCoV Ns. Some have also been reported to exhibit antiviral activity against SARS-CoV-2, HCoV-OC43, and HCoV-229E viral infections in cell culture. Our work reveals that the assembly dynamics of N condensates can be regulated by small molecules with therapeutic potential. Our approach allows for screening based on viral genome sequences alone and might enable rapid paths to drug discovery with value for confronting future pandemics.

Relationship between SARS-CoV-2 nucleocapsid protein and N gene and its application in antigen testing kits evaluation.

More than forty antigen testing kits have been approved to response the prevalence of SARS-CoV-2 and its variant strains. However, the approved antigen testing kits are not capable of quantitative detection. Here, we successfully developed a lateral flow immunoassay based on colloidal gold nanoparticles (CGNP-based LFIA) for nucleocapsid (N) protein of SARS-CoV-2 quantitative detection. Delta strain (NMDC60042793) of SARS-CoV-2 have been cultured and analyzed by our developed digital PCR and LFIA methods to explore the relationship between N protein amount and N gene level. It indicated that the linear relationship (y = 47 ×) between N protein molecule number and N gene copy number exhibited very well (R2 = 0.995), the virus titers and N protein amount can be roughly estimated according to nucleic acid testing. Additionally, detection limits (LODs) of nine approved antigen testing kits also have been evaluated according to the Guidelines for the registration review of 2019-nCoV antigen testing reagents. Only three antigen testing kits had LODs as stated in the instructions, the LODs of Kits have been converted into the N gene and N protein levels, according to the established relationships among virus titer vers. N gene and antigen. Results demonstrated that the sensitivity of nucleic acid testing is at least 1835 times higher than that of antigen testing. We expect that the relationship investigation and testing kits evaluation have the important directive significance to precise epidemic prevention.

Therapeutic potency of compound RMY-205 for pulmonary fibrosis induced by SARS-CoV-2 nucleocapsid protein.

Pulmonary fibrosis is a typical sequela of coronavirus disease 2019 (COVID-19), which is linked with a poor prognosis for COVID-19 patients. However, the underlying mechanism of pulmonary fibrosis induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unclear. Here, we demonstrated that the nucleocapsid (N) protein of SARS-CoV-2 induced pulmonary fibrosis by activating pulmonary fibroblasts. N protein interacted with the transforming growth factor ß receptor I (TßRI), to disrupt the interaction of TßRI-FK506 Binding Protein12 (FKBP12), which led to activation of TßRI to phosphorylate Smad3 and boost expression of pro-fibrotic genes and secretion of cytokines to promote pulmonary fibrosis. Furthermore, we identified a compound, RMY-205, that bound to Smad3 to disrupt TßRI-induced Smad3 activation. The therapeutic potential of RMY-205 was strengthened in mouse models of N protein-induced pulmonary fibrosis. This study highlights a signaling pathway of pulmonary fibrosis induced by N protein and demonstrates a novel therapeutic strategy for treating pulmonary fibrosis by a compound targeting Smad3.

The role of SARS-CoV-2 nucleocapsid protein in antiviral immunity and vaccine development.

ABSTRACTThe coronavirus disease 2019 (COVID-19) has caused enormous health risks and global economic disruption. This disease is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 nucleocapsid protein is a structural protein involved in viral replication and assembly. There is accumulating evidence indicating that the nucleocapsid protein is multi-functional, playing a key role in the pathogenesis of COVID-19 and antiviral immunity against SARS-CoV-2. Here, we summarize its potential application in the prevention of COVID-19, which is based on its role in inflammation, cell death, antiviral innate immunity, and antiviral adaptive immunity.

Structural insights into ribonucleoprotein dissociation by nucleocapsid protein interacting with non-structural protein 3 in SARS-CoV-2.

The coronavirus nucleocapsid (N) protein interacts with non-structural protein 3 (Nsp3) to facilitate viral RNA synthesis and stabilization. However, structural information on the N-Nsp3 complex is limited. Here, we report a 2.6 Å crystal structure of the N-terminal domain (NTD) of the N protein in complex with the ubiquitin-like domain 1 (Ubl1) of Nsp3 in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One NTD and two Ubl1s formed a stable heterotrimer. We performed mutational analysis to reveal the key residues for this interaction. We confirmed the colocalization of SARS-CoV-2 N and Nsp3 in Huh-7 cells. N-Ubl1 interaction also exists in SARS-CoV and Middle East respiratory syndrome coronavirus. We found that SARS-CoV-2 Ubl1 competes with RNA to bind N protein in a dose-dependent manner. Based on our results, we propose a model for viral ribonucleoprotein dissociation through N protein binding to Ubl1 of Nsp3.

SARS coronavirus 2-reactive antibodies in bovine colostrum.

Objective: To determine if bovine colostrum and sera have antibodies that react with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Animals: Dairy and beef cattle from North America and Europe, sampled before and after the SARS-CoV-2 pandemic. Procedures: Indirect ELISAs using whole bovine coronavirus (BCoV) and SARS-CoV-2; whole SARS-CoV-2 Spike 1, Spike 2, and nucleocapsid proteins; and SARS-CoV-2-specific nucleocapsid peptide as antigens. Virus neutralization assay for BCoV. Surrogate virus neutralization assay for SARS-CoV-2. Results: Antibodies reactive to BCoV were highly prevalent in samples collected from cattle before and after the SARS-CoV-2 pandemic. Antibodies reactive with SARS-CoV-2 were present in the same samples, and apparently increased in prevalence after the SARS-CoV-2 pandemic. These antibodies had variable reactivity with the spike and nucleocapsid proteins of SARS-CoV-2 but were apparently not specific for SARS-CoV-2. Conclusions: Bovine coronavirus continues to be endemic in cattle populations, as indicated by the high prevalence of antibodies to the virus in colostrum and serum samples. Also, the prevalent antibodies to SARS-CoV-2 in bovine samples, before and after the pandemic, are likely the result of responses to epitopes on the spike and nucleocapsid proteins that are shared between the 2 betacoronaviruses. Cross-reactive antibodies in bovine colostrum could be examined for prophylactic or therapeutic effects on SARS-CoV-2 infections in humans.

Anticorps réactifs au coronavirus du SRAS 2 dans le colostrum bovin. Objectif: Déterminer si le colostrum et des échantillons de sérum bovins contiennent des anticorps qui réagissent avec le coronavirus 2 du syndrome respiratoire aigu sévère (SRAS-CoV-2). Animaux: Bovins laitiers et bovins de boucherie d'Amérique du Nord et d'Europe, échantillonnés avant et après la pandémie de SARS-CoV-2. Procédures: Épreuves ELISA indirectes utilisant le coronavirus bovin entier (BCoV) et le SARS-CoV-2; ensemble des protéines SARS-CoV-2 Spicule 1, Spicule 2 et nucléocapside; et le peptide de nucléocapside spécifique du SARS-CoV-2 comme antigènes. Test de neutralisation du virus pour le BCoV. Virus de substitution pour le test de neutralisation du SRAS-CoV-2. Résultats: Les anticorps réactifs au BCoV étaient très répandus dans les échantillons prélevés sur les bovins avant et après la pandémie de SRAS-CoV-2. Des anticorps réactifs au SRAS-CoV-2 étaient présents dans les mêmes échantillons et leur prévalence a apparemment augmenté après la pandémie de SRAS-CoV-2. Ces anticorps avaient une réactivité variable avec les protéines de spicule et de nucléocapside du SARS-CoV-2 mais n'étaient apparemment pas spécifiques du SARS-CoV-2. Conclusion: Le coronavirus bovin continue d'être endémique dans les populations bovines, comme l'indique la forte prévalence d'anticorps dirigés contre le virus dans les échantillons de colostrum et de sérum. De plus, les anticorps prévalents contre le SRAS-CoV-2 dans les échantillons de bovins, avant et après la pandémie, sont probablement le résultat de réponses à des épitopes sur les protéines de spicule et de nucléocapside qui sont partagées entre les 2 bêtacoronavirus. Les anticorps à réaction croisée dans le colostrum bovin pourraient être examinés pour leurs effets prophylactiques ou thérapeutiques sur les infections par le SRAS-CoV-2 chez l'humain.(Traduit par Dr Serge Messier).

Detection of SARS-CoV-2 Nucleocapsid and Microvascular Disease in the Brain: A Case Report.

BACKGROUND AND OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can cause a wide range of neurologic complications; however, its neuropenetrance during the acute phase of the illness is unknown. METHODS: Extracellular vesicles were isolated from brain biopsy tissue from a patient undergoing epilepsy surgery using ultracentrifugation and analyzed by Western blot and qPCR for the presence of virus protein and RNA, respectively. Biopsy tissue was assessed by immunohistochemistry for the presence of microvascular damage and compared with 3 other non-COVID surgical epilepsy brain tissues. RESULTS: We demonstrate the presence of viral nucleocapsid protein in extracellular vesicles and microvascular disease in the brain of a patient undergoing epilepsy surgery shortly after SARS-CoV-2 infection. Endothelial cell activation was indicated by increased levels of platelet endothelial cell adhesion molecule-1 and was associated with fibrinogen leakage and immune cell infiltration in the biopsy tissue as compared with control non-COVID surgical epilepsy brain tissues. DISCUSSION: Despite the lack of evidence of viral replication within the brain, the presence of the nucleocapsid protein was associated with disease-specific endothelial cell activation, fibrinogen leakage, and immune cell infiltration.

Preparation of a new monoclonal antibody against nucleocapsid protein of swine acute diarrhea syndrome coronavirus and identification of its linear antigenic epitope.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), which causes severe diarrhea in newborn piglets, was first identified in Southern China in 2017. Since the Nucleocapsid (N) protein in SADS-CoV is highly conserved and plays a key role in virus replication, it is often used as a target protein in scientific research. In this study, the N protein of SADS-CoV was successfully expressed, and a new monoclonal antibody (mAb), 5G12, against the protein was generated successfully. The mAb 5G12 can be used to detect SADS-CoV strains by indirect immunofluorescence assay (IFA) and western blotting. The mAb 5G12 epitope was located to amino acids 11 EQAESRGRK 19 by evaluating the antibody for reactivity with a series of truncated N protein segments. The biological information analysis showed that the antigenic epitope had a high antigenic index and conservation. This study will help further understand the protein structure and function of SADS-CoV and in the establishment of specific SADS-CoV detection methods.

Digital and Analog Detection of SARS-CoV-2 Nucleocapsid Protein via an Upconversion-Linked Immunosorbent Assay.

The COVID-19 crisis requires fast and highly sensitive tests for the early stage detection of the SARS-CoV-2 virus. For detecting the nucleocapsid protein (N protein), the most abundant viral antigen, we have employed upconversion nanoparticles that emit short-wavelength light under near-infrared excitation (976 nm). The anti-Stokes emission avoids autofluorescence and light scattering and thus enables measurements without optical background interference. The sandwich upconversion-linked immunosorbent assay (ULISA) can be operated both in a conventional analog mode and in a digital mode based on counting individual immune complexes. We have investigated how different antibody combinations affect the detection of the wildtype N protein and the detection of SARS-CoV-2 (alpha variant) in lysed culture fluid via the N protein. The ULISA yielded a limit of detection (LOD) of 1.3 pg/mL (27 fM) for N protein detection independent of the analog or digital readout, which is approximately 3 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays or commercial lateral flow assays for home testing. In the case of SARS-CoV-2, the digital ULISA additionally improved the LOD by a factor of 10 compared to the analog readout.