Longitudinal neutralization activities on authentic Omicron variant provided by three doses of BBIBP-CorV vaccination during one year.

The majority of people in China have been immunized with the inactivated viral vaccine BBIBP-CorV. The emergence of the Omicron variant raised the concerns about protection efficacy of the inactivated viral vaccine in China. However, longitudinal neutralization data describing protection efficacy against Omicron variant is still lacking. Here we present one-year longitudinal neutralization data of BBIBP-CorV on authentic Omicron, Delta, and wild-type strains using 224 sera collected from 14 volunteers who have finished three doses BBIBP-CorV. The sera were also subjected for monitoring the SARS-CoV-2 specific IgG, IgA, and IgM responses on protein and peptide microarrays. The neutralization titers showed different protection efficacies against the three strains. By incorporating IgG and IgA signals of proteins and Spike protein derived peptide on microarray, panels as potential surrogate biomarkers for rapid estimation of neutralization titers were established. These data support the necessity of the 3rd dose of BBIBP-CorV vaccination. After further validation and assay development, the panels could be used for reliable, convenient and fast evaluation of the efficacy of vaccination.

Systematic profiling of SARS-CoV-2-specific IgG responses elicited by an inactivated virus vaccine identifies peptides and proteins for predicting vaccination efficacy.

One of the best ways to control COVID-19 is vaccination. Among the various SARS-CoV-2 vaccines, inactivated virus vaccines have been widely applied in China and many other countries. To understand the underlying protective mechanism of these vaccines, it is necessary to systematically analyze the humoral responses that are triggered. By utilizing a SARS-CoV-2 microarray with 21 proteins and 197 peptides that fully cover the spike protein, antibody response profiles of 59 serum samples collected from 32 volunteers immunized with the inactivated virus vaccine BBIBP-CorV were generated. For this set of samples, the microarray results correlated with the neutralization titers of the authentic virus, and two peptides (S1-5 and S2-22) were identified as potential biomarkers for assessing the effectiveness of vaccination. Moreover, by comparing immunized volunteers to convalescent and hospitalized COVID-19 patients, the N protein, NSP7, and S2-78 were identified as potential biomarkers for differentiating COVID-19 patients from individuals vaccinated with the inactivated SARS-CoV-2 vaccine. The comprehensive profile of humoral responses against the inactivated SARS-CoV-2 vaccine will facilitate a deeper understanding of the vaccine and provide potential biomarkers for inactivated virus vaccine-related applications.

Infection with novel coronavirus (SARS-CoV-2) causes pneumonia in Rhesus macaques.

The 2019 novel coronavirus (SARS-CoV-2) outbreak is a major challenge for public health. SARS-CoV-2 infection in human has a broad clinical spectrum ranging from mild to severe cases, with a mortality rate of ~6.4% worldwide (based on World Health Organization daily situation report). However, the dynamics of viral infection, replication and shedding are poorly understood. Here, we show that Rhesus macaques are susceptible to the infection by SARS-CoV-2. After intratracheal inoculation, the first peak of viral RNA was observed in oropharyngeal swabs one day post infection (1 d.p.i.), mainly from the input of the inoculation, while the second peak occurred at 5 d.p.i., which reflected on-site replication in the respiratory tract. Histopathological observation shows that SARS-CoV-2 infection can cause interstitial pneumonia in animals, characterized by hyperemia and edema, and infiltration of monocytes and lymphocytes in alveoli. We also identified SARS-CoV-2 RNA in respiratory tract tissues, including trachea, bronchus and lung; and viruses were also re-isolated from oropharyngeal swabs, bronchus and lung, respectively. Furthermore, we demonstrated that neutralizing antibodies generated from the primary infection could protect the Rhesus macaques from a second-round challenge by SARS-CoV-2. The non-human primate model that we established here provides a valuable platform to study SARS-CoV-2 pathogenesis and to evaluate candidate vaccines and therapeutics.

Gemcitabine, lycorine and oxysophoridine inhibit novel coronavirus (SARS-CoV-2) in cell culture.

The emerging SARS-CoV-2 infection associated with the outbreak of viral pneumonia in China is ongoing worldwide. There are no approved antiviral therapies to treat this viral disease. Here we examined the antiviral abilities of three broad-spectrum antiviral compounds gemcitabine, lycorine and oxysophoridine against SARS-CoV-2 in cell culture. We found that all three tested compounds inhibited viral replication in Vero-E6 cells at noncytotoxic concentrations. The antiviral effect of gemcitabine was suppressed efficiently by the cytidine nucleosides. Additionally, combination of gemcitabine with oxysophoridine had an additive antiviral effect against SARS-CoV-2. Our results demonstrate that broad-spectrum antiviral compounds may have a priority for the screening of antiviral compounds against newly emerging viruses to control viral infection.

Development of Neutralization Assay Using an eGFP Chikungunya Virus.

Chikungunya virus (CHIKV), a member of the Alphavirus genus, is an important human emerging/re-emerging pathogen. Currently, there are no effective antiviral drugs or vaccines against CHIKV infection. Herein, we construct an infectious clone of CHIKV and an eGFP reporter CHIKV (eGFP-CHIKV) with an isolated strain (assigned to Asian lineage) from CHIKV-infected patients. The eGFP-CHIKV reporter virus allows for direct visualization of viral replication through the levels of eGFP expression. Using a known CHIKV inhibitor, ribavirin, we confirmed that the eGFP-CHIKV reporter virus could be used to identify inhibitors against CHIKV. Importantly, we developed a novel and reliable eGFP-CHIKV reporter virus-based neutralization assay that could be used for rapid screening neutralizing antibodies against CHIKV.

[Bioassay for enrich-blood bioactivity of Agelicae Sinensis Radix].

Danggui, Agelicae Sinensis Radix, is a widely used Chinese herb to enrich blood, but its quality cannot be effectively assessed by the known chemical markers such as ferulic acid, ligustilide, polysaccharides, etc. A new bioassay was therefore developed to quantify the Enrich-Blood Bioactivity (EBB) for the quality assessment of Danggui raw materials. Danggui sample was first extracted with ethanol and water, respectively. Then the ethanolic extract and water extract were mixed as a test sample to quantify the amount of EBB by mice experiment. The blood deficiency mode in mice was developed by intraperitoneal injecting cyclophospharmide and phenylhdrazine hydrochloride. The quantity of red blood cell was chosen as EBB marker. Cyclosporine A was chosen as a control substance. EBB in analytes was quantified by the amount reaction of parallel line analysis (3, 3') method. The results indicated that the reliability test for quantifying EBB was passed through and the measured value was valid. The analytes showed the significant EBB (P < 0.05). The correlation coefficient was 0.9984 (n=5) between the amount of cyclosporine A (0.035-0.56 g x kg(-1)) and the increased number of red blood cell. The relative standard deviation (RSY) on the amount of EBB was estimated to be 6.15% (n = 6) by six replicated tests, and the confidence limit rate was 26.68% (n = 6). Five Danggui samples, which were collected from different cultivation areas with various morphological characters, showed the variety of EBB in the range of 21.95-44.16 U x g(-1). It is concluded that the developed method is accurate to quantify the EBB of Danggui raw materials, and is therefore suitable to assess its quality.

Quantum dot-induced viral capsid assembling in dissociation buffer.

Viruses encapsulating inorganic nanoparticles are a novel type of nanostructure with applications in biomedicine and biosensors. However, the encapsulation and assembly mechanisms of these hybridized virus-based nanoparticles (VNPs) are still unknown. In this article, it was found that quantum dots (QDs) can induce simian virus 40 (SV40) capsid assembly in dissociation buffer, where viral capsids should be disassembled. The analysis of the transmission electron microscope, dynamic light scattering, sucrose density gradient centrifugation, and cryo-electron microscopy single particle reconstruction experimental results showed that the SV40 major capsid protein 1 (VP1) can be assembled into ≈25 nm capsids in the dissociation buffer when QDs are present and that the QDs are encapsulated in the SV40 capsids. Moreover, it was determined that there is a strong affinity between QDs and the SV40 VP1 proteins (KD=2.19E-10 M), which should play an important role in QD encapsulation in the SV40 viral capsids. This study provides a new understanding of the assembly mechanism of SV40 virus-based nanoparticles with QDs, which may help in the design and construction of other similar virus-based nanoparticles.

Reversibly acetylated lysine residues play important roles in the enzymatic activity of Escherichia coli N-hydroxyarylamine O-acetyltransferase.

CobB is a bacterial NAD(+)-dependent protein deacetylase. Although progress has been made in functional studies of this protein in recent years, its substrates and biological functions are still largely unclear. Using proteome microarray technology, potential substrates of Escherichia coli CobB were screened and nine proteins were identified, including N-hydroxyarylamine O-acetyltransferase (NhoA). In vitro acetylation/deacetylation of NhoA was verified by western blotting and mass spectrometry, and two acetylated lysine residues were identified. Site-specific mutagenesis experiments showed that mutation of each acetylated lysine decreased the acetylation level of NhoA in vitro. Further analysis showed that variant NhoA proteins carrying substitutions at the two acetylated lysine residues are involved in both the O-acetyltransferase and N-acetyltransferase activity of NhoA. Structural analyses were also performed to explore the effects of the acetylated lysine residues on the activity of NhoA. These results suggest that reversible acetylation may play a role in the activity of Escherichia coli NhoA.

Rapid detection of Bacillus anthracis spores using a super-paramagnetic lateral-flow immunological detection system.

There is an urgent need for convenient, sensitive, and specific methods to detect the spores of Bacillus anthracis, the causative agent of anthrax, because of the bioterrorism threat posed by this bacterium. In this study, we firstly develop a super-paramagnetic lateral-flow immunological detection system for B. anthracis spores. This system involves the use of a portable magnetic assay reader, super-paramagnetic iron oxide particles, lateral-flow strips and two different monoclonal antibodies directed against B. anthracis spores. This detection system specifically recognises as few as 400 pure B. anthracis spores in 30 min. This system has a linear range of 4×10³-106 CFU ml⁻¹ and reproducible detection limits of 200 spores mg⁻¹ milk powder and 130 spores mg⁻¹ soil for simulated samples. In addition, this approach shows no obvious cross-reaction with other related Bacillus spores, even at high concentrations, and has no significant dependence on the duration of the storage of the immunological strips. Therefore, this super-paramagnetic lateral-flow immunological detection system is a promising tool for the rapid and sensitive detection of Bacillus anthracis spores under field conditions.

Activity-induced long-term potentiation of excitatory synapses in developing zebrafish retina in vivo.

Neural activity-induced long-term potentiation (LTP) of synaptic transmission is believed to be one of the cellular mechanisms underlying experience-dependent developmental refinement of neural circuits. Although it is well established that visual experience and neural activity are critical for the refinement of retinal circuits, whether and how LTP occurs in the retina remain unknown. Using in vivo perforated whole-cell recording and two-photon calcium imaging, we find that both repeated electrical and visual stimulations can induce LTP at excitatory synapses formed by bipolar cells on retinal ganglion cells in larval but not juvenile zebrafish. LTP induction requires the activation of postsynaptic N-methyl-D-aspartate receptors, and its expression involves arachidonic acid-dependent presynaptic changes in calcium dynamics and neurotransmitter release. Physiologically, both electrical and visual stimulation-induced LTP can enhance visual responses of retinal ganglion cells. Thus, LTP exists in developing retinae with a presynaptic locus and may serve for visual experience-dependent refinement of retinal circuits.

Multifunctional ferritin cage nanostructures for fluorescence and MR imaging of tumor cells.

Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging α(v)ß(3) integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging.

[Social-psychological factors contributing to male juvenile delinquency].

OBJECTIVE: To study the major social-psychological factors contributing to male juvenile delinquency. METHODS: One hundred and thirty-seven cases of male juvenile delinquents (delinquent group) and 145 aged-matched male students (control group) were enrolled in this case-control study. A questionnaire survey was conducted using the Adolescent Self-Rating Life Events Check List, the Coping Style Questionnaire, the Family Environment Scale-Chinese version, and the Social Support Rating Scale. RESULTS: The monovariate analysis showed that the total score and the scores of some factors of negative life events, the scores of immature coping styles and family conflicts, and the proportion of broken families in the delinquent group were significantly higher than those in the control group. In contrast, the scores of educational levels, study stress factor in the negative life events, mature coping styles, family environments and social supports were significantly lower in the delinquent group than those in the control group. The multivariate factors analysis showed that 7 variables were enrolled into the discriminatory equations, including negative life events (interpersonal relationship and healthy adaptation), self-condemn styles, family conflicts, subjective supports, objective supports, and utilization of social supports. The total accuracy of this equation was 92.2%. CONCLUSIONS: Negative life events in the interpersonal relationship and healthy adaptation, self-condemn styles, family conflicts, and weak social support system may be major social-psychological factors contributing to male juvenile delinquency.

Comparative analysis of mycobacterial NADH pyrophosphatase isoforms reveals a novel mechanism for isoniazid and ethionamide inactivation.

NADH pyrophosphatase (NudC) catalyses the hydrolysis of NAD(H) to AMP and NMN(H) [nicotinamide mononucleotide (reduced form)]. NudC multiple sequence alignment reveals that homologues from most Mycobacterium tuberculosis isolates, but not other mycobacterial species, have a polymorphism at the highly conserved residue 237. To elucidate the functional significance of this polymorphism, comparative analyses were performed using representative NudC isoforms from M. tuberculosis H37Rv (NudC(Rv)) and M. bovis BCG (NudC(BCG)). Biochemical analysis showed that the P237Q polymorphism prevents dimer formation, and results in a loss of enzymatic activity. Importantly, NudC(BCG) was found to degrade the active forms of isoniazid (INH), INH-NAD and ethionamide (ETH), ETH-NAD. Consequently, overexpression of NudC(BCG) in Mycobacterium smegmatis mc(2)155 and M. bovis BCG resulted in a high level of resistance to both INH and ETH. Further genetic studies showed that deletion of the nudC gene in M. smegmatis mc(2)155 and M. bovis BCG resulted in increased susceptibility to INH and ETH. Moreover, inactivation of NudC in both strains caused a defect in drug tolerance phenotype for both drugs in exposure assays. Taken together, these data suggest that mycobacterial NudC plays an important role in the inactivation of INH and ETH.

Quantum dot-aptamer nanoprobes for recognizing and labeling influenza A virus particles.

The fluorescence labeling of viruses is a useful technology for virus detection and imaging. By combining the excellent fluorescence properties of quantum dots (QDs) with the high affinity and specificity of aptamers, we constructed a QD-aptamer probe. The aptamer A22, against the hemagglutinin of influenza A virus, was linked to QDs, producing the QD-A22 probe. Fluorescence imaging and transmission electron microscopy showed that the QD-A22 probe could specifically recognize and label influenza A virus particles. This QD labeling technique provides a new strategy for labeling virus particles for virus detection and imaging.

Laboratory detection and diagnosis of filoviruses.

Ebola virus (EBOV) and Marburg virus (MARV), belonging to the Filoviridae family, emerged four decades ago and caused severe viral hemorrhagic fever in human and other primates. As high as 50-90% mortality, filoviruses can cause significant threats to public health. However, so far no specific and efficient vaccine has been available, nor have other treatment methods proved to be effective. It is of great importance to detect these pathogens specific, rapidly and sensitively in order to control future filovirus outbreaks. Here, recent progresses in the development of detection and diagnosis methods for EBOV and MARV are summarized.

DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection.

The rapid detection of Bacillus anthracis, the causative agent of anthrax disease, has gained much attention since the anthrax spore bioterrorism attacks in the United States in 2001. In this work, a DNA probe functionalized quartz crystal microbalance (QCM) biosensor was developed to detect B. anthracis based on the recognition of its specific DNA sequences, i.e., the 168 bp fragment of the Ba813 gene in chromosomes and the 340 bp fragment of the pag gene in plasmid pXO1. A thiol DNA probe was immobilized onto the QCM gold surface through self-assembly via Au-S bond formation to hybridize with the target ss-DNA sequence obtained by asymmetric PCR. Hybridization between the target DNA and the DNA probe resulted in an increase in mass and a decrease in the resonance frequency of the QCM biosensor. Moreover, to amplify the signal, a thiol-DNA fragment complementary to the other end of the target DNA was functionalized with gold nanoparticles. The results indicate that the DNA probe functionalized QCM biosensor could specifically recognize the target DNA fragment of B. anthracis from that of its closest species, such as Bacillus thuringiensis, and that the limit of detection (LOD) reached 3.5 × 10(2)CFU/ml of B. anthracis vegetative cells just after asymmetric PCR amplification, but without culture enrichment. The DNA probe functionalized QCM biosensor demonstrated stable, pollution-free, real-time sensing, and could find application in the rapid detection of B. anthracis.

An auto-biotinylated bifunctional protein nanowire for ultra-sensitive molecular biosensing.

In order to obtain an ultra-sensitive molecular biosensor, we designed an auto-biotinylated bifunctional protein nanowire (bFPNw) based on the self-assembly of a yeast amyloid protein, Sup35, to which protein G and a biotin acceptor peptide (BAP) were genetically fused. These auto-biotinylated bFPNws can transfer hundreds of commercially available diagnostic enzymes to an antigen-antibody complex via the biotin-avidin system, greatly enhancing the sensitivity of immune-biosensing. Compared to our previously reported seeding-induced bFPNws (Men et al., 2009), these auto-biotinylated bFPNws gave greater signal amplification, reduced non-specific binding and improved stability. The auto-biotinylated self-assembled bFPNw molecular biosensors were applied to detect Yersinia pestis (Y. pestis) F1 antigen and showed a 2000- to 4000-fold increase in sensitivity compared to traditional immunoassays, demonstrating the potential use of these self-assembling protein nanowires in biosensing.

New regulatory mechanisms for the intracellular localization and trafficking of influenza A virus NS1 protein revealed by comparative analysis of A/PR/8/34 and A/Sydney/5/97.

During influenza A virus infection, the NS1 protein is engaged in different functions in different intracellular compartments. In this study, we showed that the NS1 of A/PR/8/34 localized in different positions from that of A/Sydney/5/97 when transiently expressed in Madin-Darby canine kidney cells. Residue 221 of NS1 was identified to be a new key residue involved in the C-terminal nuclear localization signal (NLS) and nucleolar localization signal (NoLS) of NS1 from A/Sydney/5/97. Analysis of chimeric NS1 and further mutants showed that residues responsible for the binding between NS1 and the cleavage and polyadenylation specificity factor (CPSF) are correlated with the intracellular localization of transiently expressed NS1 proteins. Fluorescence loss in photobleaching imaging revealed that the NS1 protein with both functional NLSs and nuclear export signal (NES) was able to shuttle between the nucleus and cytoplasm. Drug inhibition experiments and fluorescence resonance energy transfer analysis suggested that NS1 was exported out of the cell nuclei via a Crm1-independent pathway. Moreover, it is likely that another cytoplasmic localization-related sequence exists in the NS1 protein other than the leucine-rich NES. These findings provide new insights into the mechanism of intracellular localization and trafficking of influenza A virus NS1 protein, which is important for understanding its function.

Viral coat proteins as flexible nano-building-blocks for nanoparticle encapsulation.

Viral capsid-nanoparticle hybrid structures offer new opportunities for nanobiotechnology. We previously generated virus-based nanoparticles (VNPs) of simian virus 40 (SV40) containing quantum dots (QDs) for cellular imaging. However, as an interesting issue of nano-bio interfaces, the mechanism of nanoparticle (NP) encapsulation by viral coat proteins remains unclear. Here, four kinds of QDs with the same core/shell but different surface coatings are tested for encapsulation. All the QDs can be encapsulated efficiently and there is no correlation between the encapsulation efficiency and the surface charge of the QDs. All the SV40 VNPs encapsulating differently modified QDs show similar structures, fluorescence properties, and activity in entering living cells. These results demonstrate the flexibility of SV40 major capsid protein VP1 in NP encapsulation and provide new clues to the mechanism of NP packaging by viral shells.