Corrigendum: Heterologous vaccination with inactivated vaccine and mRNA vaccine augments antibodies against both spike and nucleocapsid proteins of SARS-CoV-2: a local study in Macao.

[This corrects the article DOI: 10.3389/fimmu.2023.1131985.].

Simultaneous and sensitive detection of SARS-CoV-2 proteins spike and nucleocapsid based on long-range SERS biosensor.

BACKGROUND: Early diagnosis of SARS-CoV-2 infection is still critical to control COVID-19 outbreak. Traditional polymerase chain reaction, enzyme-linked immunosorbent assay or lateral flow immunoassay performed poorly on detection times, sample preparation process and accuracy. Surface-enhanced Raman scattering (SERS)-based detection has emerged as a powerful analytical technique, which overcomes the above limitations. However, due to the near-field effect of traditional substrate, it is difficult to monitor the binding event of aptamers with proteins. It is obvious that a novel SERS substrate thatsupportedextended and stronger electromagnetic fields was required to hold long-range effects and allow for binding event testing. RESULTS: Driven by this challenge, we reported a long-range SERS-active substrate, which was built by inserting bowtie nanoaperture arrays in a refractive-index-symmetric environment and Au mirror surfaces, for SARS-CoV-2 protein binding event detection. Then, a double-π structure aptasensor was simply designed through the hybridization of spike (S) and nucleocapsid (N) proteins aptamers, and a corresponding complementary strand. This kind of double-π structure would dissociate when targets proteins S and N existed and led to the SERS responses decreased, which established the detection basis of our system. What's more, due to two Raman labels were involved, both proteins S and N can be sensed simultaneously. Our proposed method showed improved sensitivity with a low limit of detection for multiplex detection (1.6 × 10-16 g/mL for protein S and 1.0 × 10-16 g/mL for protein N) over a wide concentration range. SIGNIFICANCE: This represents the first long-range SERS apatasensor platform for detection of S and N proteins simultaneously. Our method showed high sensitivity, selectivity, reproducibility, stability and remarkable recoveries in human in saliva and serum samples, which is particularly important for the early diagnostics of COVID as well as for future unknown coronavirus.

Nucleocapsid proteins from human coronaviruses possess phase separation capabilities and promote FUS pathological aggregation.

The nucleocapsid (N) protein is an essential structural component necessary for genomic packaging and replication in various human coronaviruses (HCoVs), such as SARS-CoV-2 and MERS-CoV. Recent studies have revealed that the SARS-CoV-2 N protein exhibits a high capacity for liquid-liquid phase separation (LLPS), which plays multiple roles in viral infection and replication. In this study, we systematically investigate the LLPS capabilities of seven homologous N proteins from different HCoVs using a high-throughput protein phase separation assay. We found that LLPS is a shared intrinsic property among these N proteins. However, the phase separation profiles of the various N protein homologs differ, and they undergo phase separation under distinct in vitro conditions. Moreover, we demonstrate that N protein homologs can co-phase separate with FUS, a SG-containing protein, and accelerate its liquid-to-solid phase transition and amyloid aggregation, which is closely related to amyotrophic lateral sclerosis. Further study shows that N protein homologs can directly bind to the low complexity domain of FUS. Together, our work demonstrates that N proteins of different HCoVs possess phase separation capabilities, which may contribute to promoting pathological aggregation of host proteins and disrupting SG homeostasis during the infection and replication of various HCoVs.

Clinical Utility of Sero-Immunological Responses Against SARS-CoV-2 Nucleocapsid Protein During Subsequent Prevalence of Wild-Type, Delta Variant, and Omicron Variant.

As nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 is immunogenic but not targeted in vaccines, it could be useful in distinguishing natural infection from vaccination. We aimed to investigate the clinical utility of sero-immunological responses against the nucleocapsid protein. Nucleocapsid antibody immunoassay study with 302 coronavirus disease 2019 (COVID-19) patients showed lower titers in immunocompromised patients (P < 0.001), higher titers in higher severity (P = 0.031), and different seroconversion rates and titers according to variants of concern. Longitudinal evaluation of nucleocapsid antibodies using 513 samples from 291 COVID-19 patients revealed that it could persist up to 556 days from symptom onset. Interferon gamma release assay against the nucleocapsid protein showed poor response, precluding the deduction of a cut-off for the nucleocapsid protein. In conclusion, nucleocapsid antibody provides instructive clues about the immunogenicity of nucleocapsid proteins by different seroconversion rates and titers according to the severity of infection, host immune status, and different variants of concern.

Heterologous vaccination with inactivated vaccine and mRNA vaccine augments antibodies against both spike and nucleocapsid proteins of SARS-CoV-2: a local study in Macao.

The mRNA vaccines (RVs) can reduce the severity and mortality of severe acute respiratory syndrome coronavirus (SARS-CoV-2). However, almost only the inactivated vaccines (IVs) but no RVs had been used in mainland China until most recently, and the relaxing of its anti-pandemic strategies in December 2022 increased concerns about new outbreaks. In comparison, many of the citizens in Macao Special Administrative Region of China received three doses of IV (3IV) or RV (3RV), or 2 doses of IV plus one booster of RV (2IV+1RV). By the end of 2022, we recruited 147 participants with various vaccinations in Macao and detected antibodies (Abs) against the spike (S) protein and nucleocapsid (N) protein of the virus as well as neutralizing antibodies (NAb) in their serum. We observed that the level of anti-S Ab or NAb was similarly high with both 3RV and 2IV+1RV but lower with 3IV. In contrast, the level of anti-N Ab was the highest with 3IV like that in convalescents, intermediate with 2IV+1RV, and the lowest with 3RV. Whereas no significant differences in the basal levels of cytokines related to T-cell activation were observed among the various vaccination groups before and after the boosters. No vaccinees reported severe adverse events. Since Macao took one of the most stringent non-pharmaceutical interventions in the world, this study possesses much higher confidence in the vaccination results than many other studies from highly infected regions. Our findings suggest that the heterologous vaccination 2IV+1RV outperforms the homologous vaccinations 3IV and 3RV as it induces not only anti-S Ab (to the level as with 3RV) but also anti-N antibodies (via the IV). It combines the advantages of both RV (to block the viral entry) and IV (to also intervene the subsequent pathological processes such as intracellular viral replication and interference with the signal transduction and hence the biological functions of host cells).

At-Home COVID-19 Rapid Antigen Test Down to 0.03 pg mL-1 of Nucleocapsid Protein.

Rapid and ultra-sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for early screening and management of COVID-19. Currently, the real-time reverse transcription polymerase chain reaction (rRT-PCR) is the primary laboratory method for diagnosing SARS-CoV-2. It is not suitable for at-home COVID-19 diagnostic test due to the long operating time, specific equipment, and professional procedures. Here an all-printed photonic crystal (PC) microarray with portable device for at-home COVID-19 rapid antigen test is reported. The fluorescence-enhanced effect of PC amplifies the fluorescence intensity of the labeled probe, achieving detection of nucleocapsid (N-) protein down to 0.03 pg mL-1 . A portable fluorescence intensity measurement instrument gives the result (negative or positive) by the color of the indicator within 5 s after inserting the reacted PC microarray test card. The N protein in inactivated virus samples (with cycle threshold values of 26.6-40.0) can be detected. The PC microarray provides a general and easy-to-use method for the timely monitoring and eventual control of the global coronavirus pandemic.

COVID-19 vaccinations in pregnancy: Save mother and baby from COVID-19 pandemic.

OBJECTIVE: The current study investigated the immune response of maternal coronavirus disease 2019 (COVID-19) vaccination and vertical transmission of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) and nucleocapsid (N) proteins. STUDY DESIGN: This retrospective study included pregnant women in Bahrain Defense Force Hospital from March 2021 to September 2021 who were vaccinated with Sinopharm or Pfizer/BioNTech. Testing of anti-N and -S levels from paired samples of maternal and umbilical cord blood was performed at the time of delivery. The immune response to vaccination, association with maternal and fetal factors, and vertical transmission of antibodies were studied. RESULTS: The current study included 79 pregnant women. The median gestational age for those vaccinated with Sinopharm was 28 weeks and those vaccinated with Pfizer was 31 weeks, with 100% of the vaccinated population generating antibodies and showing vertical transmission. The anti-N and -S titers and interval frequencies varied in both vaccinations. The anti-N and -S and transfer ratio statistically correlated with maternal age, gestational age at delivery, latency period, and birth weight of the neonates differently in both vaccines. In addition, the peak level of antibodies and transfer ratios varied. CONCLUSION: Although variations are exhibited in both types of vaccination, the vaccinated pregnant population generated a significant level of anti-N and -S and showed vertical transmission.

Expressão e purificação da proteína nucleocapsídica recombinante de SARS-CoV-2

Known as the Covid-19 Pandemic, caused by the SARS-Cov 2 virus, it became known as one of the great challenges of the 21st century and brought to industry and research the need to create defensive parameters and of rapid effectiveness for the biological study of possible threats to the health of the general population. The Sars-COV-2 Nucleocapsid protein, responsible for stabilizing the nucleocapsid, in addition to being involved in RNA synthesis and translation, and acting as an interferon antagonist and RNA interference suppressor, favoring viral replication, became the target of this study with the objective of creating parameters for its production in the laboratory in the form of recombinant protein. Aiming at high yield and good quality, through its expression in competent bacteria Escherichia coli BL21(DE3) and the improvement in the steps of its creation, counting on sonication of the expressed bacteria, its purification and protein concentration. The present study reached the objectives for the production of this protein for the Laboratório de Imunoquímica of the Instituto Butantan, also being able to guide the production of this and other recombinant proteins that end up becoming of interest for study and research in search of the health of the population.

Denominada como Pandemia do Covid-19, causada pelo vírus SARS-Cov 2, ficou conhecida como um dos grandes desafios do século XXI e trouxe à indústria e à pesquisa a necessidade de criar parâmetros defensivos e de rápida efetividade para o estudo biológico de possíveis ameaças à saúde da população em geral. A proteína Nucleocapsídica do Sars-COV-2, responsável por estabilizar o nucleocapsídeo, além de estar envolvida na síntese e tradução do RNA, e atuar como antagonista do interferon e supressor de interferência do RNA, favorecendo a replicação viral, se tornou alvo deste estudo com o objetivo de criar parâmetros para a sua produção em laboratório em forma de proteína recombinante. Visando o alto rendimento e boa qualidade, através de sua expressão em bactéria competente Escherichia coli BL21(DE3) e a melhora nos passos de sua criação, contando com sonicação das bactérias expressas, sua purificação e concentração proteica. O presente estudo alcançou os objetivos para produção dessa proteína para o Laboratório de Imunoquímica do Instituto Butantan, também sendo capaz de guiar a produção dessa e de outras proteínas recombinantes que acabam se tornando de interesse para estudo e pesquisa em busca da saúde da população.

Highly Stable Buffer-Based Zinc Oxide/Reduced Graphene Oxide Nanosurface Chemistry for Rapid Immunosensing of SARS-CoV-2 Antigens.

The widespread and long-lasting effect of the COVID-19 pandemic has called attention to the significance of technological advances in the rapid diagnosis of SARS-CoV-2 virus. This study reports the use of a highly stable buffer-based zinc oxide/reduced graphene oxide (bbZnO/rGO) nanocomposite coated on carbon screen-printed electrodes for electrochemical immuno-biosensing of SARS-CoV-2 nuelocapsid (N-) protein antigens in spiked and clinical samples. The incorporation of a salt-based (ionic) matrix for uniform dispersion of the nanomixture eliminates multistep nanomaterial synthesis on the surface of the electrode and enables a stable single-step sensor nanocoating. The immuno-biosensor provides a limit of detection of 21 fg/mL over a linear range of 1-10 000 pg/mL and exhibits a sensitivity of 32.07 ohms·mL/pg·mm2 for detection of N-protein in spiked samples. The N-protein biosensor is successful in discriminating positive and negative clinical samples within 15 min, demonstrating its proof of concept used as a COVID-19 rapid antigen test.

Biophysical analysis of SARS-CoV-2 transmission and theranostic development via N protein computational characterization.

Recently, SARS-CoV-2 has been identified as the causative factor of viral infection called COVID-19 that belongs to the zoonotic beta coronavirus family known to cause respiratory disorders or viral pneumonia, followed by an extensive attack on organs that express angiotensin-converting enzyme II (ACE2). Human transmission of this virus occurs via respiratory droplets from symptomatic and asymptomatic patients, which are released into the environment after sneezing or coughing. These droplets are capable of staying in the air as aerosols or surfaces and can be transmitted to persons through inhalation or contact with contaminated surfaces. Thus, there is an urgent need for advanced theranostic solutions to control the spread of COVID-19 infection. The development of such fit-for-purpose technologies hinges on a proper understanding of the transmission, incubation, and structural characteristics of the virus in the external environment and within the host. Hence, this article describes the development of an intrinsic model to describe the incubation characteristics of the virus under varying environmental factors. It also discusses on the evaluation of SARS-CoV-2 structural nucleocapsid protein properties via computational approaches to generate high-affinity binding probes for effective diagnosis and targeted treatment applications by specific targeting of viruses. In addition, this article provides useful insights on the transmission behavior of the virus and creates new opportunities for theranostics development.

Mechanistic differences between HIV-1 and SIV nucleocapsid proteins and cross-species HIV-1 genomic RNA recognition.

BACKGROUND: The nucleocapsid (NC) domain of HIV-1 Gag is responsible for specific recognition and packaging of genomic RNA (gRNA) into new viral particles. This occurs through specific interactions between the Gag NC domain and the Psi packaging signal in gRNA. In addition to this critical function, NC proteins are also nucleic acid (NA) chaperone proteins that facilitate NA rearrangements during reverse transcription. Although the interaction with Psi and chaperone activity of HIV-1 NC have been well characterized in vitro, little is known about simian immunodeficiency virus (SIV) NC. Non-human primates are frequently used as a platform to study retroviral infection in vivo; thus, it is important to understand underlying mechanistic differences between HIV-1 and SIV NC. RESULTS: Here, we characterize SIV NC chaperone activity for the first time. Only modest differences are observed in the ability of SIV NC to facilitate reactions that mimic the minus-strand annealing and transfer steps of reverse transcription relative to HIV-1 NC, with the latter displaying slightly higher strand transfer and annealing rates. Quantitative single molecule DNA stretching studies and dynamic light scattering experiments reveal that these differences are due to significantly increased DNA compaction energy and higher aggregation capability of HIV-1 NC relative to the SIV protein. Using salt-titration binding assays, we find that both proteins are strikingly similar in their ability to specifically interact with HIV-1 Psi RNA. In contrast, they do not demonstrate specific binding to an RNA derived from the putative SIV packaging signal. CONCLUSIONS: Based on these studies, we conclude that (1) HIV-1 NC is a slightly more efficient NA chaperone protein than SIV NC, (2) mechanistic differences between the NA interactions of highly similar retroviral NC proteins are revealed by quantitative single molecule DNA stretching, and (3) SIV NC demonstrates cross-species recognition of the HIV-1 Psi RNA packaging signal.

Detection and subtype analysis of VP1 gene of enterovirus 71 strains isolated from children with hand,foot and mouth disease in Shanghai area during the first half year of 2009 / 中华传染病杂志

Objective To understand the characteristics of molecular epidemiology of enterovirus 71(EV71) in children with hand, foot and mouth disease (HFMD) in Shanghai area during the first half year of 2009. Methods Seventy-three throat swabs and 38 stool samples were collected from 95 hospitalized children with clinical diagnosis of HFMD in Children's Hospital of Fudan University during April to May 2009. TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR) and nest RT-PCR were used to detect EV71 VP1, followed by gene sequencing analysis. Results Six of the 73 throat swabs were EV71 positive with the detection rate of 8.2%. In the 38 stool samples, 24 were EV71 positive with the detection rate of 63.2%. Twenty-eight nested RT-PCR positive samples were sequenced and the genetic analysis showed that 27 were C4 subtype,which were absolute dominant strain and the other one was C2 subtype. The isolated strain from a fatal case was C4 subtype and there was no obvious mutation found in VP1 region. Conclusions EV71 is an important pathogen in HFMD children in Shanghai area during April to May 2009. C4 subtype strains are absolutely dominant, and accompanied by epidemic strains of subtype C2.

Detection and identification of human metapneumovirus infection in Shenzhen children / 中华检验医学杂志

Objective To detect human metapneumovirus (hMPV)in respiratory intection rapidly and perform molecular analysis of hMPV.Methods Seven respiratory tract virus(11 subtypes)were assessed using multiplex PCR technology and flexible Multi-Analyte Profiling(suspension array).Human metapneumovirus was confirmed by using a real.Time reverse ranscriptase CR(RT-PCR)assay followed by sequencing.The cladogram analysis was performed further.Results The virus were detected in 40.2%(19/47)samples collected from clinicsl respiratory tract infections,including 8(42.1%)HRSV,7(36.8%)influenza virus,1(5.3%)parainfluenza virus,1(5.3%)rhinovirus,1(5.3%) coxsackievirus and 1(5.3%)human etapneumovirus infections.This is the first time that hMPV was deteced from clinical samples in Shenzhen.The sequencing of specific fragment of neucleoprotein of hMPV showed this hMPV shares over 98% homology with Beijing strain.Japan strain and Thailand strain.The cladogram analysis showed that they were in the same cluste.Conclusions Human etapneumovirus is a maior cause of children respiratory tract disease. Multiplex PCR technology and nexible Multi-Analyte Profiling were hish sensitive and high-throughput for detection of human metapneumovirus.They axe very robust and applicable in etiology analysis.

The establishment of immunochromatographic tests trips with colloidal gold for early and rapid diagnostic of hemorrhagic fever with renal syndrome / 中华检验医学杂志

Objective To establish an early,simple and rapid colloidal gold strip method for the detection of IgM against hantavirus nucleocapsid protein in patients with hemorrhagic fever with renal syndrome(HFRS).Methods Purified recombinant Hantavirus nucleoprotein(rNP)was labeled by colloidal gold particles and then sprayed and fixed on fiberglass membrane as the combination pad.Anti-Human IgM(μ-chain specific)antibody produced in goat was fixed in the detection area,and mouse antihantavirus antibody was fixed in the quality control area.Both of them were on nitrocellous membrane strip in tandem.Together with a specimen pad ahead.The conbination pad and the nitrocellous membrane were assembled into a test strip.The colloidal gold strip assay was compared with ELISA for evaluation of specificity and sensitivity.Results The colloidal gold strip tests showed positive in the serum samples from 50 cases of HFRS which was clinically diagnosed and then verified by ELISA within 10-15 minutes.Whereas 30 serum samples of healthy donors have tested negative.Conclusions Our new colloidal gold immunochromatographic test strip method was well concordant with the ELISA assay,but the former was more raoid and simole.It could be used primary medical services.

Cultured cortical astrocytes of newborn rat can be infected by Hantaviruses / 中华传染病杂志

Objective To confirm that astrocytes from cerebral cortex of newborn rat can be the target cells of Hantaan virus (HTNV)and Seoul virus (SEOV)infection and to observe changes of astrocytes after different infection time. Methods Astrocytes were prepared from cerebral cortex of newborn rat, and then infected with HTNV and SEOV. The established virus infections were confirmed by detection of virus nucleocapsid protein (NP) and S segment RNA in astrocytes using double-label immunofluoreseence, Western-blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Results The astrocytes from cerebral cortex of newborn rat were cultured successfully in vitro, which could be infected by HTNV and SEOV. The number of infected astrocytes and the virus titer in the infected astrocytes kept on increasing along with the extended infection duration. Conclusions Astrocytes from cerebral cortex of newborn rat are the target cells for HTNV and SEOV infection. Then establishment of in vitro cultured astrocytes model for Hantaviruses infection will be helpful for the study on the pathogenesis of hemorrhagic fever with renal syndrome.