Dual-mode aptamer-driven biosensing platform for ultrasensitive and mutation-resilient detection of the SARS-CoV-2 nucleocapsid protein.

Rear Window—What Can the Gut Tell Us About Long-COVID?

SARS-CoV-2's origin should be investigated worldwide for pandemic prevention

The outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has resulted in a global pandemic with an exponential growth rate. A recent study found SARS-CoV-2 nucleocapsid protein in kidney tubules of patients infected with SARS-CoV-2 (B. Diao, C.H. Wang, R.S. Wang, Z.Q. Feng, Y.J. Tan, H.M. Wang, et al.: Human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] infection [preprint posted online April 10, 2020]. medRxiv doi:10.1101/2020.03.04.20031120), providing direct evidence of kidney susceptibility to SARS-CoV-2. However, the full extent of clinical manifestations with kidney disease and prognoses in patients with the infection remains largely unknown. Because CKD affects 10%–15% of the global population, we urgently need to clarify (1) who is susceptible to kidney damage, (2) what the clinical manifestations are and how they should be diagnosed at an early stage, and (3) how the patients should be monitored during follow-up. The recent development of large-scale "omics" analytic approaches provides a large number of publicly available data sets, such as the spatial characterization of the transcriptome and proteome in different tissues of the human body (1) and expression quantitative trait loci (eQTLs) in kidney compartments (2,3). Human angiotensin-converting enzyme 2 (ACE2) has been identified as the functional receptor of SARS-CoV-2 (4). Thus, genetic analysis of the spatial distribution of human ACE2 expression and its genetic determinants in kidney provides a promising opportunity for predicting kidney involvement during SARS-CoV-2 infection at an early stage. We first determined the gene and protein expression levels of human ACE2 along with its spatial characterization in kidney compartments in The Human Protein Atlas (https://www.proteinatlas.org/) (1). Human ACE2 was highly expressed in multiple organs, including the kidney. This was consistent with the fact that, although SARS-CoV-2 infection primarily manifests as acute respiratory illness, it has also been detected in urine samples. In the kidney, human ACE2 was specifically highly expressed in tubules rather than in glomeruli (Figure 1), suggesting tubular injury as the main consequence of SARS-CoV-2 infection in the kidney. This was validated in a recent study that observed macrophage infiltration and acute tubular damage but no severe glomerular injury (B. Diao, C.H. Wang, R.S. Wang, Z.Q. Feng, Y.J. Tan, H.M. Wang, et al.: Human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] infection [preprint posted online April 10, 2020]. medRxivdoi:10.1101/2020.03.04.20031120).Figure 1.: Human ACE2 was highly stained in tubules rather than in glomeruli in human kidney tissue. These results were derived from the Human Protein Atlas database (https://www.proteinatlas.org/). Protein expression in normal kidney tissue from six patients was detected by immunohistochemistry using antibodies HPA000288 (rabbit; Sigma) and CAB026174 (mouse; R&D Systems). The ages of patients range from 16 to 70 years old. As shown, human ACE2 was not detected in glomeruli but was highly stained in tubules.We then searched for the genetic determinants of human ACE2 expression in kidney tubulointerstitial tissue to evaluate the susceptibility to kidney damage in patients infected with SARS-CoV-2. Gillies et al. (2) provided thousands of kidney-specific eQTLs in manually microdissected glomerular (n=136) and tubulointerstitial (n=166) compartments from patients with proteinuric kidney diseases, such as FSGS, minimal change disease, and membranous nephropathy (NephQTL database, http://nephqtl.org/). After searching, 49 variants with P<0.05 were detected; the lowest P value detected was 7.89×10−3, but none of the variants achieved a Bonferroni-corrected threshold P<1.02×10−3. Likewise, there were no significant eQTLs from microdissected tubulointerstitial compartments (n=119) from healthy human kidneys of white subjects undergoing surgical nephrectomy (Human Kidney eQTL Atlas, http://susztaklab.com/eqtl) (3). Therefore, the gene expression of human ACE2 in tubules is less likely to be affected by genetic variants, meaning that the general population could potentially be susceptible to consequences from SARS-CoV-2 infection on the kidney. Finally, we applied our genetic analysis pipeline to other viral infections with well known receptors (e.g., hepatitis B and D viruses). As expected, the cellular receptor of the hepatitis B and D viruses, sodium taurocholate cotransporting polypeptide, was highly specifically expressed in human hepatocytes. The strictly hepatotropic character of hepatitis B and D viruses suggests that SARS-CoV-2 may be nephrotropic. In fact, previous studies have shown a considerable degree of kidney involvement in SARS and Middle East respiratory syndrome (MERS), including AKI and kidney failure (5). Both SARS-CoV RNA and viral particles have been observed in kidney tubules from SARS autopsies (6), indicating direct infection and replication in the kidney. Dipeptidyl peptidase-4, the cellular receptor of MERS-CoV, is also expressed in kidney tubules. There is also evidence supporting kidney infection and induction of apoptosis by MERS-CoV in human ex vivo organ culture (7). The underlying mechanism of kidney involvement in patients infected with SARS-CoV-2 is still unclear, but the existing evidence suggests that the direct infection of SARS-CoV-2 in kidney tubules may play a role. It is still unclear whether the kidney may be a hidden reservoir for SARS-CoV-2 and if the virus will persistently replicate in the kidney. Thus, more attention should be given to the long-term sequelae of SARS-CoV-2 infection and intensive monitoring of kidney function seems necessary. However, it should be noted that, in contrast to the relatively high expression of the sodium taurocholate cotransporting polypeptide receptor of hepatitis B and D viruses in blood cells (normalized expression ranges from 0.1 to 2.7), the normalized expression levels of human ACE2 ranges from 0.0 to 0.3 (https://www.proteinatlas.org/), implying that SARS-CoV-2 has a relatively low viremic rate. It was reported that viral RNA was detectable by real-time PCR of plasma samples in about 15% (two of 13) of patients in the intensive-care unit and 14% (four of 28) of other patients (8). However, the clinical significance of viral RNA in plasma for affecting kidney manifestations and whether the virus itself is also present in plasma must still be determined. Thus, although SARS-CoV-2 showed nephrotropic effects, the rate of kidney injury directly caused by SARS-CoV-2 needs further evaluation. The spatial characteristics of RNA, protein expression of human ACE2, and kidney-specific eQTL analysis indicate that SARS-COV-2 could affect the kidneys of the general population infected with the virus, tubular injury might be the main pathologic manifestation of kidney involvement, and there is a necessity for intensive monitoring of kidney function during follow-up. However, because of a lack of routine screening and monitoring of kidney function, the exact incidence of kidney involvement is still unclear. Current studies have mainly focused on AKI, which is defined by increased serum creatinine or urine output criteria, with an incidence rate varying from 0.3% to 15%. Although, as mentioned above, there might be a relatively lower degree of direct kidney effects of SARS-CoV-2 compared with the effects of hepatitis B and D viruses on the liver. SARS-CoV-2–related cytokine storm and sepsis that lead to tubular and endothelial injury may also be potential pathways of kidney damage (e.g., microthrombi) (9). Thus, to determine the exact incidence rate of kidney injury due to SARS-CoV-2 infection, it is necessary to screen patients who are infected for signs of kidney damage by performing tests commonly used in the clinic, such as measuring serum creatinine or performing dipstick tests for proteinuria and hematuria. Additionally, because human ACE2 is strictly expressed in kidney tubules, the early kidney damage biomarkers such as neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, and IL-18 could also be considered for further clinical evaluation. Furthermore, because accumulated SARS-CoV-2 nucleocapsid protein was detected in kidney tubules from autopsies performed on patients with SARS-CoV-2, patients diagnosed with de novo kidney disease could be tested for virus RNA or viral antigen in urine as a possible surrogate for early diagnosis or for monitoring of disease severity. Finally, it needs to be determined whether patients with preexisting kidney disease are more susceptible to a progressive disease course or a higher risk of acute kidney failure. A global effort to better understand kidney injury due to SARS-CoV-2 is warranted. In conclusion, we provide a genetic pipeline to help explore the roadmap for kidney involvement in SARS-CoV-2 infection. This study highlights the benefits of integrating omics data sets with eQTLs to identify the roles of target genes in disease pathogenesis; this approach will also be applicable for other viruses with well known receptors. Disclosures Dr. H. Zang and Dr. Y. Zhang have nothing to disclose. Funding This study was supported by National Natural Science Foundation of China grant 81800636.

BackgroundThe coronavirus disease of 2019 (COVID-19) pandemic has led to more than 700 million confirmed cases and nearly 7 million deaths. Although Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) virus mainly infects the respiratory system, neurological complications are widely reported in both acute infection and long-COVID cases. Despite the success of vaccines and antiviral treatments, neuroinvasiveness of SARS-CoV-2 remains an important question, which is also centered on the mystery whether the virus is capable of breaching the barriers into the central nervous system.Figure 1:SARS-CoV-2 infection in the K18-hACE2 model.(A) Experimental diagram: Eight weeks-old K18-hACE2 mice were intranasally infected with SARS-CoV-2, monitored daily for symptoms, or euthanized at 5 days post-infection (DPI) to collect tissue. (B) Daily weight measurements in K18-hACE2 mice intranasally infected with SARS-CoV-2 isolate USA-WA1/2020. (C)The survival curve showing the probability of survival over 7 days after SARS-CoV-2 infection. (D) Representative mouse brain hemisphere image showing the presence of SARS-CoV-2 nucleocapsid protein by immunohistochemical staining. Boxed regions are shown on the right. Bar: 500 µm.MethodsHere using the K18-hACE2 model, we investigated the impact of acute SARS-CoV-2 infection on major parts of neurovascular systems, and found increased incidence of microhemorrhage and significant disruption of both BBB and BCSFB in K18-hACE2 mice after SARS-CoV-2 infection. Cerebral microvascular injury was accompanied by substantial pericyte damage, tight junction loss, astrogliosis, and neuroinflammation in the brain parenchyma. In addition, endothelial activation and vascular inflammation occurred at both BBB and BCSFB, as shown by upregulation of VCAM-1 and COX2 markers.Figure 2:Vascular damage and BBB breakdown in SARS-CoV-2 infected K18-hACE2 model.(A) Representative images showing IgG immunohistochemical staining in K18-hACE2 mouse brain tissues with or without SARS-CoV-2 infection. Boxed regions are shown at the bottom. (B) Representative images showing immunofluorescent staining with IgG, showing the microhemorrhage site in the cortex of K18-hACE2 mice with SARS-CoV-2 infection. Bar: 50 µm. (C) Quantification of the number of microhemorrhages per field of view in the cortex, thalamus, and hippocampus (Hipp). n= 3; ***p< 0.001; two-tailed Student’s t-test. (D) Quantification of the diameters of the microhemorrhages in the cortex, thalamus, and hippocampus. (E) Representative images showing immunofluorescent staining for IgG at the capillary level in the cortex. K18-hACE2 mice with SARS-CoV-2 infection exhibited a significant accumulation of IgG surrounding the microvessels. Bar: 50 µm. (F) Quantification of the number of leaky small blood vessels per field of view in cortex, thalamus, and hippocampus. n= 3; ***p< 0.001; two-tailed Student’s t-test. (G) Quantification of the percentage of leakage vascular area to the total area of blood vessels in cortex, thalamus, and hippocampus. n= 3.ResultsBy studying the K18-hACE2 infection model, we observed clear evidence of microvascular damage and breakdown of the blood-brain barrier (BBB). Mechanistically, SARS-CoV-2 infection caused pericyte damage, tight junction loss, endothelial activation and vascular inflammation, which together drive microvascular injury and BBB impairment.BBB tight junction loss in SARS-CoV-2 infected K18-hACE2 model.(A) The K18-hACE2 mice were used to test the BBB function after SARS-CoV-2 infection. (B) Representative images showing immunofluorescent staining for IgG, ZO-1, and Lectin in the cortex of K18-hACE2 mice. Bar: 50 µm. (C-D) Representative images showing immunofluorescent staining for ZO-1 and Lectin in the thalamus(C) and hippocampus(D) areas of K18-hACE2 mice. Bar: 50 µm. (E) Length of ZO-1-positive profiles in the cortex, thalamus, and Hipp. n= 3; ***p< 0.001; two-tailed Student’s t-test. (F-H) Representative images showing immunofluorescent staining for Claudin5 and Lectin in the cortex(F), thalamus (G), and hippocampus (H) areas of K18-hACE2 mice. Bar: 50 µm. (I) Claudin5 length in the cortex, thalamus and, Hipp. n= 3; ***p< 0.001; two-tailed Student’s t-test.ConclusionThe impact of such changes, together with astrogliosis and neuroinflammation, may drive or at least contribute to the neurological complications seen in COVID-19 patients. As SARS-CoV-2 will likely remain a major health issue for years to come, our findings provide a needed understanding of its impact on the major CNS barriers and brain homeostasis at both molecular and cellular levels.Vascular inflammation in SARS-CoV-2 infected K18-hACE2 model(A-C) Representative images showing immunofluorescent staining for VCAM1 and Lectin in the cortex, thalamus, and hippocampus of K18-hACE2 mice. Bar: 50 µm. (D) Quantification of VCAM1 and lectin signal overlap in the cortex, thalamus, and hippocampus. n= 3; ***p< 0.001; two-tailed Student’s t-test. (E-G) Representative images showing immunostaining of COX2 and Lectin in the cortex, thalamus, and hippocampus of K18-hACE2 mice. Bar: 50 µm. (H) Quantification of COX2 and Lectin signal overlap in the cortex, thalamus, and hippocampus areas of K18-hACE2 mice. n= 3; ***p< 0.001; ****p< 0.0001; two-tailed Student’s t-test. (I) Quantification of COX2 relative fluorescence intensity in the cortex, thalamus, and hippocampus of K18-hACE2 mice. n= 3; ***p< 0.001; ****p< 0.0001; two-tailed Student’s t-test.DisclosuresAll Authors: No reported disclosures

Development of nucleocapsid-specific monoclonal antibodies for SARS-CoV-2 and their ELISA diagnostics on an automatic microfluidic device

The nucleocapsid (N) protein of SARS coronavirus (SARS-CoV) is a major structural component of virions, which appears to be a multifunctional protein involved in viral RNA replication and translation. However, how N protein interacts with host protein remains largely elusive. To identify cellular proteins that interact with the N protein and to elucidate the possible involvement of N protein in SARS-Cov replication and translation, a human lung cDNA library was screened using a yeast two-hybrid system assay. In this study, we have identified Macrophage migration inhibitory factor protein (MIF) as a novel interaction partner of N protein by yeast two-hybrid system. The direct interaction and co-localization of N protein with MIF were confirmed by immunoprecipitation and confocal microscopy analysis, respectively. The mapping studies localized the critical N sequences for this interaction to amino acid 140-210 including SR motif by yeast two-hybrid system. To the best of our knowledge, this is the first report that SARS-N protein interacts with the MIF within host cells, which enhance our understanding of the molecular mechanisms of SARS replication. Key words: Severe acute respiratory syndrome (SARS) coronavirus, nucleocapsid protein, macrophage migration inhibitory factor protein, yeast two-hybrid.

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2) had led to a serious public health crisis, and no specific treatments or vaccines are available yet. A nucleocapsid protein (NP)‐based enzyme‐linked immunosorbent assay (ELISA) detection method is not only important in disease diagnosis, but is required for the evaluation of vaccine efficacy during the development of an inactivated SARS‐CoV‐2 vaccine. In this study, we expressed both the NP and N‐terminally truncated NP (ΔN‐NP) of SARS‐CoV‐2 in an Escherichia coli expression system and described the purification of the soluble recombinant NP and ΔN‐NP in details. The identities of the NP and ΔN‐NP were confirmed with mass spectrometry. We then used immunoglobulin G detection ELISAs to compare the sensitivity of NP and ΔN‐NP in detecting anti‐SARS‐CoV‐2 antibodies. ΔN‐NP showed greater sensitivity than NP in the analysis of serially diluted sera from mice and rabbits vaccinated with inactive SARS‐CoV‐2 and in human sera diluted 1:400. ΔN‐NP showed a positive detection rate similar to that of the SARS‐CoV‐2 S protein in human sera. We conclude that ΔN‐NP is a better serological marker than NP for evaluating the immunogenicity of inactivated SARS‐CoV‐2.

CORDITE: The Curated CORona Drug InTERactions Database for SARS-CoV-2

A hallmark of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the delayed interferon response. Interferons are typically produced upon host recognition of pathogen- or damage-associated molecular patterns, such as nucleic acids. While the mechanisms by which SARS-CoV-2 evades host recognition of its RNA are well studied, how it evades immune responses to cytosolic DNA-leaked from mitochondria or nuclei during infection-remains poorly understood. Here, we demonstrate that the SARS-CoV-2 nucleocapsid protein directly suppresses DNA sensing by cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS). Although primarily known for packaging the viral RNA genome, we uncover that the SARS-CoV-2 nucleocapsid protein also binds DNA with high affinity and competitively blocks cGAS activation. Using cell-free biochemical and biophysical approaches, including single-molecule optical tweezers, we show that the nucleocapsid protein binds to DNA at nanomolar concentrations and cocondenses with DNA at micromolar concentrations, thereby impeding stable cGAS-DNA interactions required for signal propagation. Hyperphosphorylation of the nucleocapsid protein diminishes its competitive binding capacity. Our findings reveal an unexpected role of the SARS-CoV-2 nucleocapsid protein in directly suppressing the cGAS-STING pathway, strongly suggesting that this contributes to the delayed interferon response during infection. This study raises the possibility that nucleocapsid proteins of other RNA viruses may also exhibit moonlighting functions by antagonizing host nucleic acid-sensing pathways.

The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of ongoing global pandemic of COVID-19, may trigger immunosuppression in the early stage and overactive immune response in the late stage of infection; However, the underlying mechanisms are not well understood. Here we demonstrated that the SARS-CoV-2 nucleocapsid (N) protein dually regulated innate immune responses, i.e., the low-dose N protein suppressed type I interferon (IFN-I) signaling and inflammatory cytokines, whereas high-dose N protein promoted IFN-I signaling and inflammatory cytokines. Mechanistically, the SARS-CoV-2 N protein dually regulated the phosphorylation and nuclear translocation of IRF3, STAT1, and STAT2. Additionally, low-dose N protein combined with TRIM25 could suppress the ubiquitination and activation of retinoic acid-inducible gene I (RIG-I). Our findings revealed a regulatory mechanism of innate immune responses by the SARS-CoV-2 N protein, which would contribute to understanding the pathogenesis of SARS-CoV-2 and other SARS-like coronaviruses, and development of more effective strategies for controlling COVID-19.

Antigenic sites on the nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) were mapped by Pepscan analysis with overlapping peptides that span the N protein sequence. Two major immunodominant epitopes located in the C-terminal region (amino acids [aa] 362 to 412) and middle region (aa 153 to 178) reacted with more than 75% of sera from SARS patients. Several minor immunodominant epitopes were reactive with about 50% of the SARS sera. Antisera from mice immunized with inactivated SARS-CoV recognized the two major immunodominant epitopes and one antigenic site located adjacent to the N-terminal region (aa 76 to 101), which did not react with the sera from SARS patients. Several monoclonal antibodies against SARS-CoV bound to the N- or C-terminal antigenic sites. These results suggest that the above antigenic sites on the N protein are important in eliciting humoral immune response against SARS-CoV in humans and animals and can be used as antigens for developing diagnostic tests.

Emerging vaccine-breakthrough severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight an urgent need for novel antiviral therapies. Understanding the pathogenesis of coronaviruses is critical for developing antiviral drugs. Here, we demonstrate that the SARS-CoV-2 N protein suppresses interferon (IFN) responses by reducing early growth response gene-1 (EGR1) expression. The overexpression of EGR1 inhibits SARS-CoV-2 replication by promoting IFN-regulated antiviral protein expression, which interacts with and degrades SARS-CoV-2 N protein via the E3 ubiquitin ligase MARCH8 and the cargo receptor NDP52. The MARCH8 mutants without ubiquitin ligase activity are no longer able to degrade SARS-CoV-2 N proteins, indicating that MARCH8 degrades SARS-CoV-2 N proteins dependent on its ubiquitin ligase activity. This study found a novel immune evasion mechanism of SARS-CoV-2 utilized by the N protein, which is helpful for understanding the pathogenesis of SARS-CoV-2 and guiding the design of new prevention strategies against the emerging coronaviruses.

This work proposed a simple and ultrasensitive nanozyme-based immunoassay on a filtration device for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP). Gold core porous platinum shell nanoparticles (Au@Pt NPs) were synthesized with high catalytic activity to oxidize 3,3',5,5'-tetramethylbenzidine, leading to an oblivious color change. The filtration device was designed based on the size difference of magnetic beads, filter membrane pore, and Au@Pt NPs. A simple, rapid, and consistent washing procedure can be performed with the help of a plastic syringe. This detection method could realize the quantitative detection of SARS-CoV-2 NP within 80 min for point-of-care needs. The limit of detection for the SARS-CoV-2 antigen was 0.01 ng/mL in buffer. The coefficients of variation of the assay were 1.78% for 10 ng/mL SARS-CoV-2 antigen, 2.03% for 1 ng/mL SARS-CoV-2 antigen, and 2.34% for the negative sample, respectively. The specificity of the detection platform was verified by the detection of various respiratory viruses. This simple and effective detection system was expected to promote substantial progress in the development and application of virus immunodetection technology.

The coronavirus disease 2019 (COVID-19) pandemic constitutes a global health emergency. Currently, there are no completely effective therapeutic medications for the management of this outbreak. The cytokine storm is a hyperinflammatory medical condition due to excessive and uncontrolled release of pro-inflammatory cytokines in patients suffering from severe COVID-19, leading to the development of acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS) and even mortality. Understanding the pathophysiology of COVID-19 can be helpful for the treatment of patients. Evidence suggests that the levels of tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1 and IL-6 are dramatically different between mild and severe patients, so they may be important contributors to the cytokine storm. Several serum markers can be predictors for the cytokine storm. This review discusses the cytokines involved in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, focusing on interferons (IFNs) and ILs, and whether they can be used in COVID-19 treatment. Moreover, we highlight several microRNAs that are involved in these cytokines and their role in the cytokine storm caused by COVID-19.

Recombinant nucleocapsid protein-based IgG enzyme-linked immunosorbent assay for the serological diagnosis of SARS